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Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice
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J Acupunct Meridian Stud 2018;--(-):--e--
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Research Article
Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice Q15
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Jadina Santos Vieira 1, Je ´ssica Aline Toreti 1, Ravena Carolina de Carvalho 1, Joa ˜o Eduardo de Arau ´jo 2, Marcelo Lourenc ¸o Silva 1, Josie Resende Torres Silva 1,* 1
Department of Physiotherapy, College of Nursing of the Federal University of AlfenasUNIFAL, Alfenas, Brazil 2 Laboratory of Neuropsychobiology and Motor Behavior, Department of Biomechanics, Medicine, and Rehabilitation of the Locomotor System, School of Medicine, University of Sa˜o Paulo, Ribeira˜o Preto, Brazil Available online - - -
Received: Jan 1, 2018 Revised: May 16, 2018 Accepted: May 25, 2018 KEYWORDS
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acupuncture-induced analgesia; inflammatory pain; neuropathic pain
Abstract The present study evaluates whether the injection of serotonin, acetylcholine, glutamate, bradykinin, histamine, or substance P (SP) into the Zusanli (Stomach 36, ST 36) acupoint can also produce the acupuncture-induced antinociceptive effect on inflammatory or neuropathic pain. In this in vivo experimental study, a total of 450 male Swiss mice were used. Mice were injected with saline or complete Freund’s adjuvant (CFA) or subjected to sham or chronic constriction injury (CCI) surgery. After the establishment of the inflammatory (4 hours) or the neuropathic pain (3 days), the animals (n Z 6) received manual acupuncture, sham acupuncture, or injection of saline, serotonin, acetylcholine, glutamate, bradykinin, histamine, or SP into the ST 36 and were evaluated for up to 24 hours. Mechanical threshold was evaluated, and the L4-L6 dorsal root ganglion was used for analysis of the transient receptor potential vanilloid type 1 overexpression. The mice from both the CFA and CCI models treated with manual acupuncture had significant increases in the thresholds for more than 24 hours. Sham acupuncture stimulation did not change the thresholds. In the mice injected with each of the mediators, the
* Corresponding author. Department of Physiotherapy, College of Nursing of the Federal University of Alfenas-UNIFAL, Avenue Jovino Fernandes Sales 2600, CEP 37130-000, Alfenas, MG, Brazil. E-mail: [email protected] (J.R.T. Silva). pISSN 2005-2901 eISSN 2093-8152 https://doi.org/10.1016/j.jams.2018.05.006 ª 2018 Medical Association of Pharmacopuncture Institute, Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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J.S. Vieira et al. thresholds were significantly increased for all times in both the CFA and CCI models. Transient receptor potential vanilloid type 1 overexpression in CFA and CCI mice was reduced at all times by injection of serotonin, acetylcholine, or SP but not by injection of glutamate, histamine, or bradykinin. Our data suggest that the neuroactive mediators released by acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia.
1. Introduction Acupuncture is an ancient treatment modality based on the theories of Traditional Chinese Medicine and involves the insertion of needles into the body followed by manual manipulation (manual acupuncture, MA) or electrical stimulation (electroacupuncture, EA) to induce its therapeutic effects, particularly for pain management [1]. Acupuncture needles come in many different gauges and lengths [2]. When acupuncture needles are inserted into the human body, the needles may go deep through (and thus injure) multiple layers, including the skin (epidermis and dermis), subcutaneous tissue (hypodermis), and muscles. Each of these layers contains special structures and cells which may respond to needling [3]. Histological studies have demonstrated that acupuncture points have several features, including high densities of nerve endings and A- and C-afferent fibers and a high concentration of neural and vascular elements, especially mast cells that can respond to stimulation [4,5]. Goldman et al [6] reported that acupuncture at the Zusanli (Stomach 36, ST 36) acupuncture point in mice significantly reduced chronic pain and increased the extracellular concentration of adenosine triphosphate and adenosine in the tissues surrounding the acupuncture points and that the local application of an adenosine A1ereceptor agonist replicated the analgesic effect of acupuncture. The needle manipulation process, through mechanical stimulation (microtrauma), can cause degranulation of the local mast cells in the target tissue and promote the release of nonneuronal mediators, including bradykinin, histamine, and substance P (SP) [5]. In addition, other nonneuronal cells, including macrophages, fibroblasts, lymphocytes, platelets, and keratinocytes, can release various transmitters (serotonin, acetylcholine, and glutamate), modulators, and inflammatory and immune factors which directly or indirectly act at the corresponding receptors on the surface of the peripheral afferent fibers to transmit the acupuncture effects [7]. Transient receptor potential vanilloid 1 (TRPV1), also known as the capsaicin receptor, shows a wide tissue distribution, and high levels of expression are observed in dorsal root ganglion (DRG). The role of acupuncture in downregulating TRPV1 after inflammatory [8] and neuropathic pain [9] was demonstrated previously. We hypothesized that acupuncture with the injection of mediators and transmitters, such as serotonin, acetylcholine, glutamate, bradykinin, histamine, and SP, can also produce the acupuncture-induced antinociceptive effect. Therefore, the aim of the present study was to determine the effects of serotonin, acetylcholine, glutamate, bradykinin, histamine, or SP injection into the ST 36 acupuncture
point on the inflammatory pain induced by complete Freund’s adjuvant (CFA) and the neuropathic pain induced by chronic constriction injury (CCI). In addition, we investigated whether the injection of mediators and transmitters into the ST 36 acupuncture point could downregulate TRPV1 after inflammatory and neuropathic pain.
2. Materials and methods 2.1. Animals This in vivo experimental study was conducted using 416 adult male Swiss mice (aged 6e7 weeks and weighing 25e35 g) from the main animal house of the Federal University of Alfenas. The animals were housed in plastic cages (27 12 17 cm, 4 per cage) at a controlled temperature (24 1 C) under a 12-hour lightedark cycle (dark cycle beginning at 7 PM), with free access to food and water. The experiment was conducted in accordance with local guidelines for animal welfare consistent with the National Research Council’s “Guide for the Care and Use of Laboratory Animals” (National Academies Press, Washington DC, USA) and after approval by the Committee of Animal Experimentation of the Federal University of Alfenas, protocol 618/2015. All possible efforts were made to minimize the number of animals used and their suffering. By Q3 compartmental analysis, the minimum number of six mice per group was shown to be adequate for statistical analysis, for which we used the software WINPEPI with a significance level of 5% and a power of 80%. In the Western blotting analysis, we used two animals for each time point, which was the minimum required to extract protein from the L4L6 DRG.
2.2. Experimental groups and procedures The animals were randomized and divided into two groups, inflammatory and neuropathic pain. a) Inflammatory pain Inflammatory pain was induced in 138 mice 4 hours after a subcutaneous injection of 20 mL of CFA (Mycobacterium tuberculosis; Sigma-Aldrich, St. Louis, MO, USA) into the plantar surface of the right paw under brief isoflurane (2% in O2) anesthesia [10]. This group was subdivided into 23 groups accordingly with the stimulation at ST 36. Saline was Q4 injected (SAL, n Z 6), and manual acupuncture (MA, n Z 6) and sham needle stimulation 1 mm below ST 36 (OUT, n Z 6) were performed; six mediators were injected in
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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ST 36-injected Mediators Affect Pain in Mice
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three doses (serotonin, acetylcholine, glutamate, SP, bradykinin, or histamine at 0.03, 0.3, and 3 mg diluted in 20 mL of CFA, n Z 6 each group). In addition, two mediators were injected in one higher dose because lower doses had no effects (bradykinin or histamine at 30 mg diluted in 20 mL of CFA, n Z 6 each group). b) Neuropathic pain Neuropathic pain was induced in 126 mice. Under anesthesia (by mask inhalation of isoflurane vaporized at concentration of 2%), an incision in the skin and femoral muscle was made to expose the three branches of the sciatic nerve. Tight ligation of one-third to half of the dorsal portion of the sciatic nerve with a 5.0 suture was performed, and the muscle and the skin were sutured after the procedure by the same person (J.S.V.) according to the study by Bennett and Xie [11]. The animals were considered neuropathic after 72 hours of CCI surgery. This group was subdivided into 21 groups accordingly with the stimulation at ST 36. Saline was injected (SAL, n Z 6), and manual acupuncture (MA, n Z 6) and sham needle stimulation 1 mm below ST 36 were performed (OUT, n Z 6); six mediators were injected in three doses (serotonin, acetylcholine, glutamate, SP, bradykinin, or histamine at 0.03, 0.3, and 3 mg diluted in 20 mL of CFA, n Z 6 each). The paw mechanical sensitivity was evaluated before the intraplantar injection of CFA or CCI (that is, baseline 1, BL1) on day 1 after 4 hours for the mice that received the CFA injection or on day 3 for the mice subjected to CCI (baseline 2, BL2). Injections of the neuroactive mediators, SAL, OUT, or MA, were given by gently restraining the mice, and all mice were then revaluated w10 minutes after the procedures (T0) and at 1 (T1), 6 (T6), and 24 (T24) hours later. The mice were observed carefully and did not exhibit any signs of stress or discomfort during the procedure.
2.3. Acupuncture point stimulation Serotonin hydrochloride, acetylcholine chloride, L-glutamic acid monosodium salt hydrate, histamine (SigmaAldrich), bradykinin, and SP (Tocris Bioscience, Minneapolis, MN, USA) were diluted and subsequently injected at doses of 0.03, 0.3, 3, or 30 mg as indicated. Briefly, the mediators were dissolved in a 20 mL volume of saline and administered subcutaneously into the ST 36 acupuncture point, located 5 mm below and lateral to the anterior tubercle of the tibia. For the MA, a small stainless-steel acupuncture needle, 0.18 8 mm (Dong Bang, China), was gently inserted to a depth of 1.5 mm at the ST 36 acupuncture point. The needle was slowly rotated, with reinforcing and reducing twirling manipulation, every 5 minutes for a total of 30 minutes during the acupuncture session [6,12]. The sham acupuncture point group (OUT) received acupuncture needle insertion at a nonacupuncture point located 5 mm below the ST 36. MA and OUT were performed by an acupuncturist with a certificate by the Federal Physical Therapy Council and Brazilian Society of Physical Therapists and Acupuncturists and 6 years of experience with acupuncture in animal models.
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2.4. Paw mechanical sensitivity Mechanical sensitivity was measured by using an electronic von Frey device (Insight Equipamentos, Ribeira ˜o Preto, SP, Brazil). The mice were placed in a wire chamber, in which they remained until they exhibited brief exploratory behavior (w15 minutes). The electronic pressure transducer contacted the plantar surface of the hind paw through a disposable polypropylene tip. A reduction in the threshold for mechanical stimuli was indicated by a nociceptive response (paw withdrawal) after the application of a lower applied force (in grams) [9]. The investigators responsible for mechanical threshold measurement and analysis were blinded to the experimental group assignments.
2.5. Western blotting analysis For the Western blotting analysis, we used two animals for each time point for each 16 described groups. For CFA and CCI models, we made nine stimulations (SAL, MA, OUT, serotonin, acetylcholine, glutamate, SP, bradykinin, or histamine) at four times points (T0, T1, T6, and T24 hours). In addition, we evaluated the naı¨ve (n Z 2), CFA (n Z 2, after 4 hours of paw injection), or CCI (n Z 2, 72 hours after surgery) animals. A total of 152 mice were anesthetized (by mask inhalation of isoflurane vaporized at concentration of 2%), decapitated, and exsanguinated. After laminectomy used to facilitate DRG access, the L4-6 DRG was removed [13]. The DRGs were homogenized in a lysis buffer containing protease inhibitors (Sigma-Aldrich). The protein samples were separated on a 10-well SDS/PAGE Q6 Q7 gel and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK), followed by incubation overnight at 2e8 C with a primary antibody against TRPV1 (1:500, 35 kDa; Calbiochem, Oncogene Research Products, La Jolla, CA, USA) in filtered TBS-T buffer containing 5% milk powder. The membranes were Q8 then incubated for 1 hour at room temperature with an HRP-conjugated secondary antibody (1:2000; Jackson Immuno Research Labs, West Grove, PA, USA). The blots Q9 were developed using an ECL solution (Amersham Pharmacia Biotech), and the signals were quantified with a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) [8,9,14].
2.6. Statistical analysis For the in vivo experiments, the data are reported as Q10 the means SEM. The data were analyzed using the software SPSS (version 21.0 for Windows, IBM Corp., Armonk, NY, USA), using a two-way analysis of variance (ANOVA) to compare the groups at all times. The factors analyzed were treatment, time, and the time-bytreatment interaction, and Bonferroni’s test was used for each time point. For the Western blotting analysis, the differences between the groups were evaluated using ANOVA (one-way ANOVA) followed by Bonferroni’s test. The differences were considered statistically significant at p < 0.05.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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3. Results 3.1. The changes induced by neuroactive mediators injected at the ST 36 acupuncture point in inflammatory pain The effects produced by MA or injections at ST 36 on the mechanical threshold of CFA-inflamed mice are shown in Fig. 1. A significant reduction in the threshold for the inflamed paw was observed for more than 24 hours after
J.S. Vieira et al. the CFA stimulation in the saline-injected animals. The mice treated with MA demonstrated a significant increase in the thresholds compared with the saline-treated animals for more than 24 hours after the CFA stimulation. The thresholds of the OUT group did not change. The thresholds of the mice injected with serotonin at all doses (0.03, 0.3, and 3 mg; Fig. 1A), acetylcholine at 0.03 mg (Fig. 1B), glutamate at all doses (Fig. 1C), bradykinin and histamine at 30 mg (Fig. 1D and E, respectively), and SP at 0.03 mg (Fig. 1F) were significantly greater than those of the salineinjected group for all times. The thresholds increased only
Figure 1 The effect of manual acupuncture (MA) or the injection of saline or neuroactive mediators at the ST 36 acupuncture Q13 point on the inflammatory mechanical threshold. The experiment was conducted before (BL1) and after (BL2) intraplantar injection of complete Freund’s adjuvant (CFA). The mechanical paw-withdrawal latency was measured on the inflamed hind paws 10 minutes after (T0) injection (arrow head) and subsequently at T1, T6, and T24 hours later. The bars show the means SEM of six mice per group. (*) p < 0.05 compared with the CFA/saline group. (#) p < 0.05 compared with all groups. Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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ST 36-injected Mediators Affect Pain in Mice at T6 and T24 for SP. The values were significantly different for the treatments (F5,30 Z 163.20, p Z 0.025; 115.50, p Z 0.031; 414.80, p Z 0.017; and 46.44, p Z 0.041 for Fig. 1AeC, and F, respectively; F6,36 Z 39.24, p Z 0.013 and 63.32, p Z 0.038 for Fig. 1D and E, respectively) and time (F5,150 Z 350.20, p Z 0.041; 159.30, p Z 0.039; 359.10, p Z 0.008; and 247.00, p Z 0.016 for Fig. 1AeC, and F, respectively; F5,175 Z 239.50, p Z 0.023 and 268.70, p Z 0.004 for Fig. 1D and E, respectively). Treatment time interactions were also observed (F25,150 Z 10.55, p Z 0.013; 5.21, p Z 0.032; 21.44, p Z 0.022; and 5.71, p Z 0.015 for Fig. 1AeC, and F, respectively; F30,175 Z 4.50, p Z 0.009 and 5.54, p Z 0.014 for Fig. 1D and E, respectively).
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3.2. The changes induced by neuroactive mediators injected at the ST 36 acupuncture point in neuropathic pain The effects produced by MA or injections at the ST 36 on the mechanical threshold of CCI-neuropathic mice are shown in Fig. 2. A significant reduction in the threshold for the constricted paw was observed for over 24 hours after the CCI in the saline-injected animals. The mice treated with MA had significantly increased thresholds compared with the saline-treated animals for more than 24 hours after the CCI. The thresholds for the OUT group did not change. The thresholds for the mice injected with
Figure 2 The effect of manual acupuncture (MA) or the injection or saline or neuroactive mediators at the ST 36 acupuncture point on the inflammatory mechanical threshold. The experiment was conducted before (BL1) and after (BL2) chronic constriction injury (CCI). The mechanical paw-withdrawal latency was measured on the neuropathic hind paws 10 minutes after (T0) injection (arrow head) and at various times, T1, T6, and T24 hours later. The bars show the means SEM of six mice per group. (*) p < 0.05 compared with the CCI/saline group.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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6 serotonin, acetylcholine, glutamate, bradykinin, or histamine showed significant increases at all doses compared with the saline-injected group at all times (Fig. 2AeE, respectively). SP at all doses (Fig. 2F) significantly increased the thresholds but only after T1. The values were significantly different for the treatments (F5,30 Z 29.65, p Z 0.0002; 28.17, p Z 0.0032; 19.51, p Z 0.0057; 26.34, p Z 0.0034; 28.27, p Z 0.0021; and 21.34, p Z 0.018 for Fig. 2AeF) and time (F5,150 Z 116.00, p Z 0.0006; 110.30, p Z 0.0009; 115.09, p Z 0.0018; 124.00, p Z 0.035; 148.01, p Z 0.003; and 119.10, p Z 0.027 for Fig. 2AeF). Treatment time interactions were also observed (F25,150 Z 4.28, p Z 0.003; 3.73, p Z 0.0045; 4.10, p Z 0.0017; 4.67, p Z 0.0021; 4.92, p Z 0.0039; and 3.62, p Z 0.0037 for Fig. 2AeF).
3.3. Neuroactive mediators injected at the ST 36 acupuncture point reduce TRPV1 expression in the DRG To explore the TRPV1 involvement in the effects of neuroactive mediators injected at the ST 36 acupuncture point on persistent inflammatory and neuropathic hyperalgesia, the expression of TRPV1 in the DRG was evaluated. The TRPV1 expression levels peaked at 4 hour after CFA injection (T0) and were maintained for 24 hour (T24, salineinjected mice, Fig. 3A), which coincided with the mechanical nociceptive threshold. Supporting the behavioral findings, MA stimulation or the injection of serotonin, acetylcholine, bradykinin, or SP, but not the injection of glutamate or histamine, downregulated the TRPV1 receptor overexpression in the DRG (F33,203 Z 30.20, p Z 0.004). The TRPV1 expression levels peaked at 72 hour after CCI surgery (T0) and were maintained for more than 24 hour (T24, saline-injected mice, Fig. 3B). The increased expression of the TRPV1 protein was also downregulated in the CCI-neuropathic mice after MA stimulation or the injection of serotonin, acetylcholine, or SP. Interestingly, in persistent neuropathic hyperalgesia, the effectiveness of glutamate, bradykinin, or histamine treatment was not confirmed by the evaluation of the TRPV1 protein expression (F33,203 Z 17.64, p Z 0.002).
4. Discussion The present study demonstrated that neuroactive mediators injected at the ST 36 significantly decreased inflammatory and neuropathic pain. Moreover, the injection of serotonin, acetylcholine, bradykinin, or SP, but not the injection of glutamate or histamine, downregulated the CFA-induced TRPV1 expression in the DRG. In contrast, serotonin, acetylcholine, or SP, but not glutamate, bradykinin, or histamine, downregulated the CCI-induced TRPV1 expression in the DRG. Consistent with the previously reported results [6,15,16], our data demonstrated that the stimulation of the ST 36 produced a significant inhibition of CFA- and CCIinduced mechanical hyperalgesia. Injection of chemical irritants, such as bee venom [17], formalin [18], and CFA [19], into the ST 36 has also been
J.S. Vieira et al. shown to elicit persistent discharges from the primary afferent fibers (mainly C or A delta fibers) and dorsal horn neurons, which are cells with wide dynamic ranges. Therefore, needling of an acupuncture point produces the stimulation of small-diameter nerve fibers, which are crucial for producing the therapeutic effects of acupuncture. In the present study, both the inflammatory and the neuropathic pain groups had reduced mechanical thresholds after injection of serotonin at the ST 36 compared with the controls. The endogenous serotonin system could play a role in nociception because it has various functional tissuespecific receptors [20]. Peripherally, serotonin triggers excitation and sensitization of primary nociceptive afferent fibers as well as the nociceptive neurons from which these fibers originate in the DRG. This contributes to peripheral sensitization, hyperalgesia [21], and peristaltic, secretory, vasodilatory, vagal, and nociceptive reflexes in the gastrointestinal tract [22,23]. In the central nervous system, low doses of serotonin injected via the intrathecal route are known to exert antinociceptive effects, whereas larger doses induce a pronociceptive effect. The underlying mechanisms for these effects are still not completely understood [24]. In the central nervous system, serotonin can be released via descending pain pathways to modulate nociceptive signaling in the spinal cord. Evidence suggests that the levels of serotonin increase in the spinal cord and that the uptake of its precursor (5-hydroxytryptophan; [25]) or a selective inhibitor of serotonin (fluoxetine; [26]) results in enhanced analgesia. Acupuncture stimulation has been reported to markedly increase the density of local mastocytes and activated mastocyte degranulation at the acupuncture points [7,10]. Therefore, the serotonin released by platelets and mast cells after acupunctureinduced tissue injury stimulates peptidergic afferent nerve fibers or enhances their excitability and contributes peripherally to acupuncture-induced analgesia [7]. In this experiment, both inflammatory pain and neuropathic pain were reduced by acetylcholine injection at the ST 36. When central cholinergic receptors are activated, the analgesic effect of acupuncture significantly increases [26,27], and intrathecal atropine blocked the relieving effects of EA [28]. Peripherally, acetylcholine is a major excitatory neurotransmitter that produces various effects on sensory perception and pain control [27]. Ferreira and Nakamura Q11 [29] described a peripheral analgesic effect of cholinergic agents and suggested that cholinergic agents may cause analgesia by increasing cyclic GMP at the nociceptor level. In addition, human keratinocytes synthesize, secrete, and degrade nonneuronal acetylcholine [30]. Therefore, our results suggest that acupuncture-induced tissue injury may enhance the release of acetylcholine by keratinocytes, which acts locally and can participate in acupunctureinduced analgesia. We observed that an injection of glutamate at the ST 36 reduced the mechanical thresholds for both inflammatory and neuropathic pain. Increased glutamate levels in the skin have been found in response to inflammatory agents, which results in the depolarization of primary afferent C fibers and in pain-related behavior. Accordingly, tissuedamaging stimuli would release glutamate and lead to
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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Figure 3 TRPV1 expression in the DRG after CFA-induced inflammatory pain (A) and CCI-induced neuropathic pain (B) and manual Q14 acupuncture (MA) or the injection of saline or neuroactive mediators at the ST 36 acupuncture point. (*) p < 0.05 for the MA, OUT, or the injected groups compared with the CFA/saline group (A) or the CCI/saline group (B). ACH Z acetylcholine; BRAD Z bradykinin; CCI Z chronic constriction injury; CFA Z complete Freund’s adjuvant; DRG Z dorsal root ganglion; GLUT Z glutamine; HIS Z histamine; SAL Z saline; SP Z substance P; TRPV1 Z transient receptor potential vanilloid 1.
increased C fiber activation and to subsequent paininduced behavior [31]. Although peripheral glutamate injection may cause allodynia or hyperalgesia in the localized area at the injection site [32], the present findings suggest that the peripheral glutamate released from acupunctureinduced tissue injury may contribute to acupunctureinduced analgesia. The injection of histamine into the ST 36 produced antinociceptive effects on both inflammatory and neuropathic pain. The effects of histamine can be either analgesic or algesic, depending on the site of action and the dose [33,34]. Histamine in the central nervous system is
found in both histaminergic neurons and in mast cells. Peripherally, histamine is stored in mast cells and other types of cells [35]. In rat and mouse skin, H3 receptors are present in Ad sensory fibers associated with arterioles in the deep dermis and seem to act directly on peripheral fibers in the skin to reduce peptide-mediated inflammation and nociception [36]. Taken together, our data suggest that the peripheral histamine released from mast cells after acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia. Finally, acupuncture can increase SP and the calcitonin geneerelated peptide in peripheral nerve terminals and in
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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8 blood in humans and rats [37]. In this experiment, both inflammatory pain and neuropathic pain were reduced by the injection of SP at the ST 36 acupuncture point. Although SP provokes vasodilation, its local application did not induce itching or pain sensations even at concentrations 50- to 1000-fold above the vasodilatory threshold, suggesting that vasoactive concentrations of SP do not excite nociceptors [38]. In addition, SP not only contributes to local vasodilatation but also induces histamine release from the adjacent mast cells, which produces a flare and further activates other sensory nerve endings [39]. Similarly, histamine has been shown to mediate the release of SP and glutamate in inflammatory conditions [40]. Together with histamine, other mast cellederived mediators (such as serotonin, adenosine, bradykinin, and SP) can sensitize the nociceptor TRPV1, which is abundantly expressed in various anatomical layers of the ST 36 and is involved in the acupunctureinduced analgesic effect [41]. Our results show that TRPV1 expression is affected by the injection of neuroactive mediators at the ST 36. Peripheral synaptic transmission from the DRG neurons to the spinal cord dorsal horn is crucial for pain signaling. Our results show that CFA- and CCI-induced pain reliably induces mechanical and thermal pain, which is accompanied by an increase in TRPV1. The injection of serotonin, acetylcholine, bradykinin, or SP, but not the injection of glutamate or histamine, downregulated the CFA-induced TRPV1 expression, and injection of serotonin, acetylcholine, or SP, but not glutamate, bradykinin, or histamine, downregulated the CCI-induced TRPV1 expression in the DRG. Similar results in the DRG were found for EA in CFAinduced inflammatory pain [8] and a fibromyalgia model [42] and for moxibustion in visceral pain in bone marrow cells [43]. We suggest that the mechanism that underlies the injection at the ST 36 may be the inhibition of protein kinase A activation, which results in the downregulation of TRPV1 and reduction of sensitization. Moreover, it is likely that the effects of injection at the ST 36 on inflammatory and neuropathic pain may involve multiple systems beyond the local effect. Zhang et al [44] demonstrated that EA pretreatment at the Gallbladder 20 (GB20) and Triple Heater 5 (TE5) acupuncture points upregulates CB1 receptor expression in the trigeminal ganglion and inhibits the changes in interleukin-1b, cyclooxygenase 2, calcitonin geneerelated peptide, and prostaglandin E2 in a rodent model of migraine. Xu et al [45] suggested that immune responses and metabolic processes may be involved in mediating the effects of moxibustion at the ST 36 and Bladder 23 (BL23) and that peroxiredoxin I and the inositol 1,4,5-triphosphate receptors may be the potential targets in rats with collageninduced arthritis. Acupuncture has the potential to produce analgesic effects in various neuropathic pain models. Recently, Zhao et al [46] demonstrated that EA at Governing Vessel acupuncture points (GV6 and GV9) had a positive impact in a spinal cord injury model in rats. This effect is related to the neuroprotective effect of neurotrophin-3, which may increase the polarization of M2 microglia/macrophages. EA stimulation alleviates spinal nerve ligationeinduced neuropathic pain, at least in part through the inhibition of spinal glial activation
J.S. Vieira et al. [47]. EA also alleviates the muscle atrophy induced by sciatic nerve injection injury by upregulating agrin and epsilonacetylcholine receptors and downregulating gammaacetylcholine receptors [48]. Moreover, the use of lowfrequency transcutaneous electrical acupuncture point stimulation attenuates CCI-induced neuropathic pain, and its analgesic effect is likely mediated by the upregulation of opioid receptor expression in the DRG [49]. Taken together, these results indicate that there are specific peripheral (neural, skeletal muscle, connective tissue, and/or immunological) and central (opioid, cannabinoid and serotonin release, and receptor upregulation/ downregulation) effects of acupuncture that may be the key components to successful therapy and that to obtain such effects, the stimulation technique and the target tissue must be adapted to the condition treated. One limitation inherent in this study concerns the way the doses were determined. The functions of mediators are severalfold. Some of them activate neurons directly (e.g., the application of bradykinin, serotonin, or glutamate directly evokes action potentials), and/or they sensitize neurons for mechanical, thermal, and chemical stimuli (e.g., bradykinin and prostaglandins increase the excitability of neurons so that mechanical stimuli evoke action potentials at a lower threshold than under control conditions). In response to peripheral injury, many neuroactive mediators are released from blood cells, vascular endothelial cells, immune cells (including mast cells), and nerve fibers [50]. We selected the lowest dose of injection as 0.03 mg for all mediators and used 10- and 100-fold higher doses (0.3 mg and 3 mg). When the maximum dose did not change the withdrawal thresholds, the dose was increased to 30 mg (bradykinin and histamine). We suggest that these doses are related to the doses released when acupuncture is administered into the skin, but additional experimentation and/or binding studies are required to define the exact doses of injected mediators. In summary, our data are consistent with the concept that the neuroactive mediators released during acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia. These results may lead to clinical studies of pharmacopuncture application to acupuncture points for pain management. Additional experimentation is required to define the effects of neuroactive mediators on the afferent neurons in acupuncture-induced analgesia.
Financial support This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´gico (CNPq) Grant No. 447340/2014-0. J.S.V. received a Master’s Level Fellowship supported by the Coordenac ¸˜ ao de Aperfeic ¸oamento de Q16 Pessoal de Nı´vel Superior (CAPES).
Acknowledgments The authors are grateful for the excellent technical support from Luciana Costa Teodoro and Ze ´lia de Fa ´tima Fernandes.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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References
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[1] Patil S, Sen S, Bral M, Reddy S, Bradley KK, Cornett EM, et al. The role of acupuncture in pain management. Curr Pain Headache Rep 2016;20(4):22. [2] Wang G, Gao Q, Li J, Tian Y, Hou J. Impact of needle diameter on long-term dry needling treatment of chronic lumbar myofascial pain syndrome. Am J Phys Med Rehabil 2016;95(7): 483e94. [3] Ahn AC, Martinsen OG. Electrical characterization of acupuncture points: technical issues and challenges. J Altern Complement Med 2007;13(8):817e24. [4] Li AH, Zhang JM, Xie YK. Human acupuncture points mapped in rats are associated with excitable muscle/skin-nerve complexes with enriched nerve endings. Brain Res 2004;1012(1-2): 154e9. [5] Chen B, Li MY, Guo Y, Zhao X, Lim HC. Mast cell-derived exosomes at the stimulated acupoints activating the neuroimmuno regulation. Chin J Integr Med 2016. [6] 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(7):883e8. [7] Zhang D, Spielmann A, Wang L, Ding G, Huang F, Gu Q, et al. Mast-cell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2. Physiol Res 2012;61(1):113e24. [8] Chen W-H, Tzen JTC, Hsieh CL, Chen YH, Lin T-J, Chen S-Y, et al. Attenuation of TRPV1 and TRPV4 expression and function in mouse inflammatory pain models using electroacupuncture. Evid-Based Complement Altern Med: eCAM 2012;2012:636848. [9] Jiang Y-L, Yin X-H, Shen Y-F, He X-F, Fang J-Q. Low frequency electroacupuncture alleviated spinal nerve ligation induced mechanical allodynia by inhibiting TRPV1 upregulation in ipsilateral undamaged dorsal root ganglia in rats. Evid-Based Complement Altern Med: eCAM 2013;2013:170910. [10] Li WM, Cui KM, Li N, Gu QB, Schwarz W, Ding GH, et al. Analgesic effect of electroacupuncture on complete Freund’s adjuvant-induced inflammatory pain in mice: a model of antipain treatment by acupuncture in mice. Jpn J Physiol 2005;55(6):339e44. [11] Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33(1):87e107. [12] Yu Z, Luo L, Li Y, Wu Q, Deng S, Lian S, et al. Different manual manipulations and electrical parameters exert different therapeutic effects of acupuncture. J Tradit Chin Med 2014; 34(6):754e8. [13] Unger MD, Maus TP, Puffer RC, Newman LK, Currier BL, Beutler AS. Laminotomy for lumbar dorsal root ganglion access and injection in swine. J Vis Exp 2017;(128). [14] Sant’Anna MB, Kusuda R, Bozzo TA, Bassi GS, Alves-Filho JC, Cunha FQ, et al. Medial plantar nerve ligation as a novel model of neuropathic pain in mice: pharmacological and molecular characterization. Sci Rep 2016;6:26955. [15] Kim SK, Park JH, Bae SJ, Kim JH, Hwang BG, Min BI, et al. Effects of electroacupuncture on cold allodynia in a rat model of neuropathic pain: mediation by spinal adrenergic and serotonergic receptors. Exp Neurol 2005;195(2): 430e6. [16] Lu KW, Hsu CK, Hsieh CL, Yang J, Lin YW. Probing the effects and mechanisms of electroacupuncture at ipsilateral or contralateral ST36-ST37 acupoints on CFA-induced inflammatory pain. Sci Rep 2016;6:22123. [17] Baek YH, Huh JE, Lee JD, Choi DY, Park DS. Antinociceptive effect and the mechanism of bee venom acupuncture (Apipuncture) on inflammatory pain in the rat model of collagen-
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
induced arthritis: mediation by alpha2-Adrenoceptors. Brain Res 2006;1073e1074:305e10. Silva ML, Silva JR, Prado WA. The antinociceptive effect of electroacupuncture at different depths of acupoints and under the needling surface. Chin Med 2012;7:3. Chen HS, Qu F, He X, Liao D, Kang SM, Lu SJ. The antinociceptive effect and the possible mechanism of acupoint stimulation caused by chemical irritants in the bee venom pain model. Brain Res 2010;1355:61e9. Viguier F, Michot B, Hamon M, Bourgoin S. Multiple roles of serotonin in pain control mechanismseimplications of 5-HT(7) and other 5-HT receptor types. Eur J Pharmacol 2013;716(13):8e16. Sommer C. Serotonin in pain and analgesia. Mol Neurobiol 2004;30(2):117e25. Zhu X, Liu Z, Niu W, Wang Y, Zhang A, Qu H, et al. Effects of electroacupuncture at ST25 and BL25 in a Sennae-induced rat model of diarrhoea-predominant irritable bowel syndrome. Acupunct Med 2017;35(3):216e23. Zhu X, Liu Z, Qu H, Niu W, Gao L, Wang Y, et al. The effect and mechanism of electroacupuncture at LI11 and ST37 on constipation in a rat model. Acupunct Med 2016;34(3): 194e200. Kayser V, Latremoliere A, Hamon M, Bourgoin S. N-methyl-Daspartate receptor-mediated modulations of the antiallodynic effects of 5-HT1B/1D receptor stimulation in a rat model of trigeminal neuropathic pain. Eur J Pain 2011;15(5): 451e8. Chang FC, Tsai HY, Yu MC, Yi PL, Lin JG. The central serotonergic system mediates the analgesic effect of electroacupuncture on ZUSANLI (ST36) acupoints. J Biomed Sci 2004; 11(2):179e85. da Silva JR, da Silva ML, Prado WA. Electroacupuncture at 2/100 hz activates antinociceptive spinal mechanisms different from those activated by electroacupuncture at 2 and 100 hz in responder rats. Evid Based Complement Alternat Med 2013;2013:205316. Wang J, Liu J, Chen S, Gao Y, Meng F, Qiao L. Acupuncture effects on the hippocampal cholinergic system in a rat model of neuropathic pain. Neural Regen Res 2012;7(3):212e8. Silva JR, Silva ML, Prado WA. Analgesia induced by 2- or 100-Hz electroacupuncture in the rat tail-flick test depends on the activation of different descending pain inhibitory mechanisms. J Pain 2011;12(1):51e60. Ferreira SH, Nakamura M. II - Prostaglandin hyperalgesia: the peripheral analgesic activity of morphine, enkephalins and opioid antagonists. Prostaglandins 1979;18(2):191e200. Grando SA, Kist DA, Qi M, Dahl MV. Human keratinocytes synthesize, secrete, and degrade acetylcholine. J Invest Dermatol 1993;101(1):32e6. Carlton SM. Nociceptive primary afferents: they have a mind of their own. J Physiol 2014;592(16):3403e11. Honda K, Noma N, Shinoda M, Miyamoto M, Katagiri A, Kita D, et al. Involvement of peripheral ionotropic glutamate receptors in orofacial thermal hyperalgesia in rats. Mol Pain 2011;7:75. Farzin D, Asghari L, Nowrouzi M. Rodent antinociception following acute treatment with different histamine receptor agonists and antagonists. Pharmacol Biochem Behav 2002; 72(3):751e60. Mojtahedin A, Tamaddonfard E, Zanboori A. Antinociception induced by central administration of histamine in the formalin test in rats. Indian J Physiol Pharmacol 2008;52(3):249e54. Cannon KE, Chazot PL, Hann V, Shenton F, Hough LB, Rice FL. Immunohistochemical localization of histamine H3 receptors in rodent skin, dorsal root ganglia, superior cervical ganglia, and spinal cord: potential antinociceptive targets. Pain 2007; 129(1-2):76e92.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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10 [36] Poveda R, Fernandez-Duenas V, Fernandez A, Sanchez S, Puig MM, Planas E. Synergistic interaction between fentanyl and the histamine H3 receptor agonist R-(alpha)-methylhistamine, on the inhibition of nociception and plasma extravasation in mice. Eur J Pharmacol 2006;541(1-2):53e6. [37] Carlsson CP, Sundler F, Wallengren J. Cutaneous innervation before and after one treatment period of acupuncture. Br J Dermatol 2006;155(5):970e6. [38] Weidner C, Klede M, Rukwied R, Lischetzki G, Neisius U, Skov PS, et al. Acute effects of substance P and calcitonin gene-related peptide in human skinea microdialysis study. J Invest Dermatol 2000;115(6):1015e20. [39] Rosa AC, Fantozzi R. The role of histamine in neurogenic inflammation. Br J Pharmacol 2013;170(1):38e45. [40] Riedel W, Neeck G. Nociception, pain, and antinociception: current concepts. Z Rheumatol 2001;60(6):404e15. [41] Wu SY, Chen WH, Hsieh CL, Lin YW. Abundant expression and functional participation of TRPV1 at Zusanli acupoint (ST36) in mice: mechanosensitive TRPV1 as an “acupuncture-responding channel”. BMC Complement Altern Med 2014;14:96. [42] Lin JG, Hsieh CL, Lin YW. Analgesic effect of electroacupuncture in a mouse fibromyalgia model: roles of TRPV1, TRPV4, and pERK. PLoS One 2015;10(6), e0128037. [43] Zou W, Lin H, Liu W, Yang B, Wu L, Duan L, et al. Moxibustion relieves visceral hyperalgesia via inhibition of transient receptor potential vanilloid 1 (TRPV1) and heat shock protein (HSP) 70 expression in rat bone marrow cells. Acupunct Med 2016;34(2):114e9.
J.S. Vieira et al. [44] Zhang H, He S, Hu Y, Zheng H. Antagonism of cannabinoid receptor 1 attenuates the anti-inflammatory effects of electroacupuncture in a rodent model of migraine. Acupunct Med 2016;34(6):463e70. [45] Xu X, Wang MM, Sun ZL, Zhou DP, Wang L, Wang FQ, et al. Discovery of serum proteomic biomarkers for prediction of response to moxibustion treatment in rats with collageninduced arthritis: an exploratory analysis. Acupunct Med 2016;34(3):184e93. [46] Zhao J, Wang L, Li Y. Electroacupuncture alleviates the inflammatory response via effects on M1 and M2 macrophages after spinal cord injury. Acupunct Med 2017;35(3):224e30. [47] Liang Y, Qiu Y, Du J, Liu J, Fang J, Zhu J, et al. Inhibition of spinal microglia and astrocytes contributes to the antiallodynic effect of electroacupuncture in neuropathic pain induced by spinal nerve ligation. Acupunct Med 2016;34(1): 40e7. [48] Yu J, Wang M, Liu J, Zhang X, Yang S. Effect of electroacupuncture on the expression of agrin and acetylcholine receptor subtypes in rats with tibialis anterior muscular atrophy induced by sciatic nerve injection injury. Acupunct Med 2017; 35(4):268e75. [49] Yu X, Zhang F, Chen B. Effect of transcutaneous electrical acupuncture point stimulation at different frequencies in a rat model of neuropathic pain. Acupunct Med 2017;35(2):142e7. [50] Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 2009;139(2):267e84.
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Research Article
Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice Q15
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Jadina Santos Vieira 1, Je ´ssica Aline Toreti 1, Ravena Carolina de Carvalho 1, Joa ˜o Eduardo de Arau ´jo 2, Marcelo Lourenc ¸o Silva 1, Josie Resende Torres Silva 1,* 1
Department of Physiotherapy, College of Nursing of the Federal University of AlfenasUNIFAL, Alfenas, Brazil 2 Laboratory of Neuropsychobiology and Motor Behavior, Department of Biomechanics, Medicine, and Rehabilitation of the Locomotor System, School of Medicine, University of Sa˜o Paulo, Ribeira˜o Preto, Brazil Available online - - -
Received: Jan 1, 2018 Revised: May 16, 2018 Accepted: May 25, 2018 KEYWORDS
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acupuncture-induced analgesia; inflammatory pain; neuropathic pain
Abstract The present study evaluates whether the injection of serotonin, acetylcholine, glutamate, bradykinin, histamine, or substance P (SP) into the Zusanli (Stomach 36, ST 36) acupoint can also produce the acupuncture-induced antinociceptive effect on inflammatory or neuropathic pain. In this in vivo experimental study, a total of 450 male Swiss mice were used. Mice were injected with saline or complete Freund’s adjuvant (CFA) or subjected to sham or chronic constriction injury (CCI) surgery. After the establishment of the inflammatory (4 hours) or the neuropathic pain (3 days), the animals (n Z 6) received manual acupuncture, sham acupuncture, or injection of saline, serotonin, acetylcholine, glutamate, bradykinin, histamine, or SP into the ST 36 and were evaluated for up to 24 hours. Mechanical threshold was evaluated, and the L4-L6 dorsal root ganglion was used for analysis of the transient receptor potential vanilloid type 1 overexpression. The mice from both the CFA and CCI models treated with manual acupuncture had significant increases in the thresholds for more than 24 hours. Sham acupuncture stimulation did not change the thresholds. In the mice injected with each of the mediators, the
* Corresponding author. Department of Physiotherapy, College of Nursing of the Federal University of Alfenas-UNIFAL, Avenue Jovino Fernandes Sales 2600, CEP 37130-000, Alfenas, MG, Brazil. E-mail: [email protected] (J.R.T. Silva). pISSN 2005-2901 eISSN 2093-8152 https://doi.org/10.1016/j.jams.2018.05.006 ª 2018 Medical Association of Pharmacopuncture Institute, Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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J.S. Vieira et al. thresholds were significantly increased for all times in both the CFA and CCI models. Transient receptor potential vanilloid type 1 overexpression in CFA and CCI mice was reduced at all times by injection of serotonin, acetylcholine, or SP but not by injection of glutamate, histamine, or bradykinin. Our data suggest that the neuroactive mediators released by acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia.
1. Introduction Acupuncture is an ancient treatment modality based on the theories of Traditional Chinese Medicine and involves the insertion of needles into the body followed by manual manipulation (manual acupuncture, MA) or electrical stimulation (electroacupuncture, EA) to induce its therapeutic effects, particularly for pain management [1]. Acupuncture needles come in many different gauges and lengths [2]. When acupuncture needles are inserted into the human body, the needles may go deep through (and thus injure) multiple layers, including the skin (epidermis and dermis), subcutaneous tissue (hypodermis), and muscles. Each of these layers contains special structures and cells which may respond to needling [3]. Histological studies have demonstrated that acupuncture points have several features, including high densities of nerve endings and A- and C-afferent fibers and a high concentration of neural and vascular elements, especially mast cells that can respond to stimulation [4,5]. Goldman et al [6] reported that acupuncture at the Zusanli (Stomach 36, ST 36) acupuncture point in mice significantly reduced chronic pain and increased the extracellular concentration of adenosine triphosphate and adenosine in the tissues surrounding the acupuncture points and that the local application of an adenosine A1ereceptor agonist replicated the analgesic effect of acupuncture. The needle manipulation process, through mechanical stimulation (microtrauma), can cause degranulation of the local mast cells in the target tissue and promote the release of nonneuronal mediators, including bradykinin, histamine, and substance P (SP) [5]. In addition, other nonneuronal cells, including macrophages, fibroblasts, lymphocytes, platelets, and keratinocytes, can release various transmitters (serotonin, acetylcholine, and glutamate), modulators, and inflammatory and immune factors which directly or indirectly act at the corresponding receptors on the surface of the peripheral afferent fibers to transmit the acupuncture effects [7]. Transient receptor potential vanilloid 1 (TRPV1), also known as the capsaicin receptor, shows a wide tissue distribution, and high levels of expression are observed in dorsal root ganglion (DRG). The role of acupuncture in downregulating TRPV1 after inflammatory [8] and neuropathic pain [9] was demonstrated previously. We hypothesized that acupuncture with the injection of mediators and transmitters, such as serotonin, acetylcholine, glutamate, bradykinin, histamine, and SP, can also produce the acupuncture-induced antinociceptive effect. Therefore, the aim of the present study was to determine the effects of serotonin, acetylcholine, glutamate, bradykinin, histamine, or SP injection into the ST 36 acupuncture
point on the inflammatory pain induced by complete Freund’s adjuvant (CFA) and the neuropathic pain induced by chronic constriction injury (CCI). In addition, we investigated whether the injection of mediators and transmitters into the ST 36 acupuncture point could downregulate TRPV1 after inflammatory and neuropathic pain.
2. Materials and methods 2.1. Animals This in vivo experimental study was conducted using 416 adult male Swiss mice (aged 6e7 weeks and weighing 25e35 g) from the main animal house of the Federal University of Alfenas. The animals were housed in plastic cages (27 12 17 cm, 4 per cage) at a controlled temperature (24 1 C) under a 12-hour lightedark cycle (dark cycle beginning at 7 PM), with free access to food and water. The experiment was conducted in accordance with local guidelines for animal welfare consistent with the National Research Council’s “Guide for the Care and Use of Laboratory Animals” (National Academies Press, Washington DC, USA) and after approval by the Committee of Animal Experimentation of the Federal University of Alfenas, protocol 618/2015. All possible efforts were made to minimize the number of animals used and their suffering. By Q3 compartmental analysis, the minimum number of six mice per group was shown to be adequate for statistical analysis, for which we used the software WINPEPI with a significance level of 5% and a power of 80%. In the Western blotting analysis, we used two animals for each time point, which was the minimum required to extract protein from the L4L6 DRG.
2.2. Experimental groups and procedures The animals were randomized and divided into two groups, inflammatory and neuropathic pain. a) Inflammatory pain Inflammatory pain was induced in 138 mice 4 hours after a subcutaneous injection of 20 mL of CFA (Mycobacterium tuberculosis; Sigma-Aldrich, St. Louis, MO, USA) into the plantar surface of the right paw under brief isoflurane (2% in O2) anesthesia [10]. This group was subdivided into 23 groups accordingly with the stimulation at ST 36. Saline was Q4 injected (SAL, n Z 6), and manual acupuncture (MA, n Z 6) and sham needle stimulation 1 mm below ST 36 (OUT, n Z 6) were performed; six mediators were injected in
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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three doses (serotonin, acetylcholine, glutamate, SP, bradykinin, or histamine at 0.03, 0.3, and 3 mg diluted in 20 mL of CFA, n Z 6 each group). In addition, two mediators were injected in one higher dose because lower doses had no effects (bradykinin or histamine at 30 mg diluted in 20 mL of CFA, n Z 6 each group). b) Neuropathic pain Neuropathic pain was induced in 126 mice. Under anesthesia (by mask inhalation of isoflurane vaporized at concentration of 2%), an incision in the skin and femoral muscle was made to expose the three branches of the sciatic nerve. Tight ligation of one-third to half of the dorsal portion of the sciatic nerve with a 5.0 suture was performed, and the muscle and the skin were sutured after the procedure by the same person (J.S.V.) according to the study by Bennett and Xie [11]. The animals were considered neuropathic after 72 hours of CCI surgery. This group was subdivided into 21 groups accordingly with the stimulation at ST 36. Saline was injected (SAL, n Z 6), and manual acupuncture (MA, n Z 6) and sham needle stimulation 1 mm below ST 36 were performed (OUT, n Z 6); six mediators were injected in three doses (serotonin, acetylcholine, glutamate, SP, bradykinin, or histamine at 0.03, 0.3, and 3 mg diluted in 20 mL of CFA, n Z 6 each). The paw mechanical sensitivity was evaluated before the intraplantar injection of CFA or CCI (that is, baseline 1, BL1) on day 1 after 4 hours for the mice that received the CFA injection or on day 3 for the mice subjected to CCI (baseline 2, BL2). Injections of the neuroactive mediators, SAL, OUT, or MA, were given by gently restraining the mice, and all mice were then revaluated w10 minutes after the procedures (T0) and at 1 (T1), 6 (T6), and 24 (T24) hours later. The mice were observed carefully and did not exhibit any signs of stress or discomfort during the procedure.
2.3. Acupuncture point stimulation Serotonin hydrochloride, acetylcholine chloride, L-glutamic acid monosodium salt hydrate, histamine (SigmaAldrich), bradykinin, and SP (Tocris Bioscience, Minneapolis, MN, USA) were diluted and subsequently injected at doses of 0.03, 0.3, 3, or 30 mg as indicated. Briefly, the mediators were dissolved in a 20 mL volume of saline and administered subcutaneously into the ST 36 acupuncture point, located 5 mm below and lateral to the anterior tubercle of the tibia. For the MA, a small stainless-steel acupuncture needle, 0.18 8 mm (Dong Bang, China), was gently inserted to a depth of 1.5 mm at the ST 36 acupuncture point. The needle was slowly rotated, with reinforcing and reducing twirling manipulation, every 5 minutes for a total of 30 minutes during the acupuncture session [6,12]. The sham acupuncture point group (OUT) received acupuncture needle insertion at a nonacupuncture point located 5 mm below the ST 36. MA and OUT were performed by an acupuncturist with a certificate by the Federal Physical Therapy Council and Brazilian Society of Physical Therapists and Acupuncturists and 6 years of experience with acupuncture in animal models.
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2.4. Paw mechanical sensitivity Mechanical sensitivity was measured by using an electronic von Frey device (Insight Equipamentos, Ribeira ˜o Preto, SP, Brazil). The mice were placed in a wire chamber, in which they remained until they exhibited brief exploratory behavior (w15 minutes). The electronic pressure transducer contacted the plantar surface of the hind paw through a disposable polypropylene tip. A reduction in the threshold for mechanical stimuli was indicated by a nociceptive response (paw withdrawal) after the application of a lower applied force (in grams) [9]. The investigators responsible for mechanical threshold measurement and analysis were blinded to the experimental group assignments.
2.5. Western blotting analysis For the Western blotting analysis, we used two animals for each time point for each 16 described groups. For CFA and CCI models, we made nine stimulations (SAL, MA, OUT, serotonin, acetylcholine, glutamate, SP, bradykinin, or histamine) at four times points (T0, T1, T6, and T24 hours). In addition, we evaluated the naı¨ve (n Z 2), CFA (n Z 2, after 4 hours of paw injection), or CCI (n Z 2, 72 hours after surgery) animals. A total of 152 mice were anesthetized (by mask inhalation of isoflurane vaporized at concentration of 2%), decapitated, and exsanguinated. After laminectomy used to facilitate DRG access, the L4-6 DRG was removed [13]. The DRGs were homogenized in a lysis buffer containing protease inhibitors (Sigma-Aldrich). The protein samples were separated on a 10-well SDS/PAGE Q6 Q7 gel and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK), followed by incubation overnight at 2e8 C with a primary antibody against TRPV1 (1:500, 35 kDa; Calbiochem, Oncogene Research Products, La Jolla, CA, USA) in filtered TBS-T buffer containing 5% milk powder. The membranes were Q8 then incubated for 1 hour at room temperature with an HRP-conjugated secondary antibody (1:2000; Jackson Immuno Research Labs, West Grove, PA, USA). The blots Q9 were developed using an ECL solution (Amersham Pharmacia Biotech), and the signals were quantified with a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) [8,9,14].
2.6. Statistical analysis For the in vivo experiments, the data are reported as Q10 the means SEM. The data were analyzed using the software SPSS (version 21.0 for Windows, IBM Corp., Armonk, NY, USA), using a two-way analysis of variance (ANOVA) to compare the groups at all times. The factors analyzed were treatment, time, and the time-bytreatment interaction, and Bonferroni’s test was used for each time point. For the Western blotting analysis, the differences between the groups were evaluated using ANOVA (one-way ANOVA) followed by Bonferroni’s test. The differences were considered statistically significant at p < 0.05.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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3. Results 3.1. The changes induced by neuroactive mediators injected at the ST 36 acupuncture point in inflammatory pain The effects produced by MA or injections at ST 36 on the mechanical threshold of CFA-inflamed mice are shown in Fig. 1. A significant reduction in the threshold for the inflamed paw was observed for more than 24 hours after
J.S. Vieira et al. the CFA stimulation in the saline-injected animals. The mice treated with MA demonstrated a significant increase in the thresholds compared with the saline-treated animals for more than 24 hours after the CFA stimulation. The thresholds of the OUT group did not change. The thresholds of the mice injected with serotonin at all doses (0.03, 0.3, and 3 mg; Fig. 1A), acetylcholine at 0.03 mg (Fig. 1B), glutamate at all doses (Fig. 1C), bradykinin and histamine at 30 mg (Fig. 1D and E, respectively), and SP at 0.03 mg (Fig. 1F) were significantly greater than those of the salineinjected group for all times. The thresholds increased only
Figure 1 The effect of manual acupuncture (MA) or the injection of saline or neuroactive mediators at the ST 36 acupuncture Q13 point on the inflammatory mechanical threshold. The experiment was conducted before (BL1) and after (BL2) intraplantar injection of complete Freund’s adjuvant (CFA). The mechanical paw-withdrawal latency was measured on the inflamed hind paws 10 minutes after (T0) injection (arrow head) and subsequently at T1, T6, and T24 hours later. The bars show the means SEM of six mice per group. (*) p < 0.05 compared with the CFA/saline group. (#) p < 0.05 compared with all groups. Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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ST 36-injected Mediators Affect Pain in Mice at T6 and T24 for SP. The values were significantly different for the treatments (F5,30 Z 163.20, p Z 0.025; 115.50, p Z 0.031; 414.80, p Z 0.017; and 46.44, p Z 0.041 for Fig. 1AeC, and F, respectively; F6,36 Z 39.24, p Z 0.013 and 63.32, p Z 0.038 for Fig. 1D and E, respectively) and time (F5,150 Z 350.20, p Z 0.041; 159.30, p Z 0.039; 359.10, p Z 0.008; and 247.00, p Z 0.016 for Fig. 1AeC, and F, respectively; F5,175 Z 239.50, p Z 0.023 and 268.70, p Z 0.004 for Fig. 1D and E, respectively). Treatment time interactions were also observed (F25,150 Z 10.55, p Z 0.013; 5.21, p Z 0.032; 21.44, p Z 0.022; and 5.71, p Z 0.015 for Fig. 1AeC, and F, respectively; F30,175 Z 4.50, p Z 0.009 and 5.54, p Z 0.014 for Fig. 1D and E, respectively).
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3.2. The changes induced by neuroactive mediators injected at the ST 36 acupuncture point in neuropathic pain The effects produced by MA or injections at the ST 36 on the mechanical threshold of CCI-neuropathic mice are shown in Fig. 2. A significant reduction in the threshold for the constricted paw was observed for over 24 hours after the CCI in the saline-injected animals. The mice treated with MA had significantly increased thresholds compared with the saline-treated animals for more than 24 hours after the CCI. The thresholds for the OUT group did not change. The thresholds for the mice injected with
Figure 2 The effect of manual acupuncture (MA) or the injection or saline or neuroactive mediators at the ST 36 acupuncture point on the inflammatory mechanical threshold. The experiment was conducted before (BL1) and after (BL2) chronic constriction injury (CCI). The mechanical paw-withdrawal latency was measured on the neuropathic hind paws 10 minutes after (T0) injection (arrow head) and at various times, T1, T6, and T24 hours later. The bars show the means SEM of six mice per group. (*) p < 0.05 compared with the CCI/saline group.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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6 serotonin, acetylcholine, glutamate, bradykinin, or histamine showed significant increases at all doses compared with the saline-injected group at all times (Fig. 2AeE, respectively). SP at all doses (Fig. 2F) significantly increased the thresholds but only after T1. The values were significantly different for the treatments (F5,30 Z 29.65, p Z 0.0002; 28.17, p Z 0.0032; 19.51, p Z 0.0057; 26.34, p Z 0.0034; 28.27, p Z 0.0021; and 21.34, p Z 0.018 for Fig. 2AeF) and time (F5,150 Z 116.00, p Z 0.0006; 110.30, p Z 0.0009; 115.09, p Z 0.0018; 124.00, p Z 0.035; 148.01, p Z 0.003; and 119.10, p Z 0.027 for Fig. 2AeF). Treatment time interactions were also observed (F25,150 Z 4.28, p Z 0.003; 3.73, p Z 0.0045; 4.10, p Z 0.0017; 4.67, p Z 0.0021; 4.92, p Z 0.0039; and 3.62, p Z 0.0037 for Fig. 2AeF).
3.3. Neuroactive mediators injected at the ST 36 acupuncture point reduce TRPV1 expression in the DRG To explore the TRPV1 involvement in the effects of neuroactive mediators injected at the ST 36 acupuncture point on persistent inflammatory and neuropathic hyperalgesia, the expression of TRPV1 in the DRG was evaluated. The TRPV1 expression levels peaked at 4 hour after CFA injection (T0) and were maintained for 24 hour (T24, salineinjected mice, Fig. 3A), which coincided with the mechanical nociceptive threshold. Supporting the behavioral findings, MA stimulation or the injection of serotonin, acetylcholine, bradykinin, or SP, but not the injection of glutamate or histamine, downregulated the TRPV1 receptor overexpression in the DRG (F33,203 Z 30.20, p Z 0.004). The TRPV1 expression levels peaked at 72 hour after CCI surgery (T0) and were maintained for more than 24 hour (T24, saline-injected mice, Fig. 3B). The increased expression of the TRPV1 protein was also downregulated in the CCI-neuropathic mice after MA stimulation or the injection of serotonin, acetylcholine, or SP. Interestingly, in persistent neuropathic hyperalgesia, the effectiveness of glutamate, bradykinin, or histamine treatment was not confirmed by the evaluation of the TRPV1 protein expression (F33,203 Z 17.64, p Z 0.002).
4. Discussion The present study demonstrated that neuroactive mediators injected at the ST 36 significantly decreased inflammatory and neuropathic pain. Moreover, the injection of serotonin, acetylcholine, bradykinin, or SP, but not the injection of glutamate or histamine, downregulated the CFA-induced TRPV1 expression in the DRG. In contrast, serotonin, acetylcholine, or SP, but not glutamate, bradykinin, or histamine, downregulated the CCI-induced TRPV1 expression in the DRG. Consistent with the previously reported results [6,15,16], our data demonstrated that the stimulation of the ST 36 produced a significant inhibition of CFA- and CCIinduced mechanical hyperalgesia. Injection of chemical irritants, such as bee venom [17], formalin [18], and CFA [19], into the ST 36 has also been
J.S. Vieira et al. shown to elicit persistent discharges from the primary afferent fibers (mainly C or A delta fibers) and dorsal horn neurons, which are cells with wide dynamic ranges. Therefore, needling of an acupuncture point produces the stimulation of small-diameter nerve fibers, which are crucial for producing the therapeutic effects of acupuncture. In the present study, both the inflammatory and the neuropathic pain groups had reduced mechanical thresholds after injection of serotonin at the ST 36 compared with the controls. The endogenous serotonin system could play a role in nociception because it has various functional tissuespecific receptors [20]. Peripherally, serotonin triggers excitation and sensitization of primary nociceptive afferent fibers as well as the nociceptive neurons from which these fibers originate in the DRG. This contributes to peripheral sensitization, hyperalgesia [21], and peristaltic, secretory, vasodilatory, vagal, and nociceptive reflexes in the gastrointestinal tract [22,23]. In the central nervous system, low doses of serotonin injected via the intrathecal route are known to exert antinociceptive effects, whereas larger doses induce a pronociceptive effect. The underlying mechanisms for these effects are still not completely understood [24]. In the central nervous system, serotonin can be released via descending pain pathways to modulate nociceptive signaling in the spinal cord. Evidence suggests that the levels of serotonin increase in the spinal cord and that the uptake of its precursor (5-hydroxytryptophan; [25]) or a selective inhibitor of serotonin (fluoxetine; [26]) results in enhanced analgesia. Acupuncture stimulation has been reported to markedly increase the density of local mastocytes and activated mastocyte degranulation at the acupuncture points [7,10]. Therefore, the serotonin released by platelets and mast cells after acupunctureinduced tissue injury stimulates peptidergic afferent nerve fibers or enhances their excitability and contributes peripherally to acupuncture-induced analgesia [7]. In this experiment, both inflammatory pain and neuropathic pain were reduced by acetylcholine injection at the ST 36. When central cholinergic receptors are activated, the analgesic effect of acupuncture significantly increases [26,27], and intrathecal atropine blocked the relieving effects of EA [28]. Peripherally, acetylcholine is a major excitatory neurotransmitter that produces various effects on sensory perception and pain control [27]. Ferreira and Nakamura Q11 [29] described a peripheral analgesic effect of cholinergic agents and suggested that cholinergic agents may cause analgesia by increasing cyclic GMP at the nociceptor level. In addition, human keratinocytes synthesize, secrete, and degrade nonneuronal acetylcholine [30]. Therefore, our results suggest that acupuncture-induced tissue injury may enhance the release of acetylcholine by keratinocytes, which acts locally and can participate in acupunctureinduced analgesia. We observed that an injection of glutamate at the ST 36 reduced the mechanical thresholds for both inflammatory and neuropathic pain. Increased glutamate levels in the skin have been found in response to inflammatory agents, which results in the depolarization of primary afferent C fibers and in pain-related behavior. Accordingly, tissuedamaging stimuli would release glutamate and lead to
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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Figure 3 TRPV1 expression in the DRG after CFA-induced inflammatory pain (A) and CCI-induced neuropathic pain (B) and manual Q14 acupuncture (MA) or the injection of saline or neuroactive mediators at the ST 36 acupuncture point. (*) p < 0.05 for the MA, OUT, or the injected groups compared with the CFA/saline group (A) or the CCI/saline group (B). ACH Z acetylcholine; BRAD Z bradykinin; CCI Z chronic constriction injury; CFA Z complete Freund’s adjuvant; DRG Z dorsal root ganglion; GLUT Z glutamine; HIS Z histamine; SAL Z saline; SP Z substance P; TRPV1 Z transient receptor potential vanilloid 1.
increased C fiber activation and to subsequent paininduced behavior [31]. Although peripheral glutamate injection may cause allodynia or hyperalgesia in the localized area at the injection site [32], the present findings suggest that the peripheral glutamate released from acupunctureinduced tissue injury may contribute to acupunctureinduced analgesia. The injection of histamine into the ST 36 produced antinociceptive effects on both inflammatory and neuropathic pain. The effects of histamine can be either analgesic or algesic, depending on the site of action and the dose [33,34]. Histamine in the central nervous system is
found in both histaminergic neurons and in mast cells. Peripherally, histamine is stored in mast cells and other types of cells [35]. In rat and mouse skin, H3 receptors are present in Ad sensory fibers associated with arterioles in the deep dermis and seem to act directly on peripheral fibers in the skin to reduce peptide-mediated inflammation and nociception [36]. Taken together, our data suggest that the peripheral histamine released from mast cells after acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia. Finally, acupuncture can increase SP and the calcitonin geneerelated peptide in peripheral nerve terminals and in
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8 blood in humans and rats [37]. In this experiment, both inflammatory pain and neuropathic pain were reduced by the injection of SP at the ST 36 acupuncture point. Although SP provokes vasodilation, its local application did not induce itching or pain sensations even at concentrations 50- to 1000-fold above the vasodilatory threshold, suggesting that vasoactive concentrations of SP do not excite nociceptors [38]. In addition, SP not only contributes to local vasodilatation but also induces histamine release from the adjacent mast cells, which produces a flare and further activates other sensory nerve endings [39]. Similarly, histamine has been shown to mediate the release of SP and glutamate in inflammatory conditions [40]. Together with histamine, other mast cellederived mediators (such as serotonin, adenosine, bradykinin, and SP) can sensitize the nociceptor TRPV1, which is abundantly expressed in various anatomical layers of the ST 36 and is involved in the acupunctureinduced analgesic effect [41]. Our results show that TRPV1 expression is affected by the injection of neuroactive mediators at the ST 36. Peripheral synaptic transmission from the DRG neurons to the spinal cord dorsal horn is crucial for pain signaling. Our results show that CFA- and CCI-induced pain reliably induces mechanical and thermal pain, which is accompanied by an increase in TRPV1. The injection of serotonin, acetylcholine, bradykinin, or SP, but not the injection of glutamate or histamine, downregulated the CFA-induced TRPV1 expression, and injection of serotonin, acetylcholine, or SP, but not glutamate, bradykinin, or histamine, downregulated the CCI-induced TRPV1 expression in the DRG. Similar results in the DRG were found for EA in CFAinduced inflammatory pain [8] and a fibromyalgia model [42] and for moxibustion in visceral pain in bone marrow cells [43]. We suggest that the mechanism that underlies the injection at the ST 36 may be the inhibition of protein kinase A activation, which results in the downregulation of TRPV1 and reduction of sensitization. Moreover, it is likely that the effects of injection at the ST 36 on inflammatory and neuropathic pain may involve multiple systems beyond the local effect. Zhang et al [44] demonstrated that EA pretreatment at the Gallbladder 20 (GB20) and Triple Heater 5 (TE5) acupuncture points upregulates CB1 receptor expression in the trigeminal ganglion and inhibits the changes in interleukin-1b, cyclooxygenase 2, calcitonin geneerelated peptide, and prostaglandin E2 in a rodent model of migraine. Xu et al [45] suggested that immune responses and metabolic processes may be involved in mediating the effects of moxibustion at the ST 36 and Bladder 23 (BL23) and that peroxiredoxin I and the inositol 1,4,5-triphosphate receptors may be the potential targets in rats with collageninduced arthritis. Acupuncture has the potential to produce analgesic effects in various neuropathic pain models. Recently, Zhao et al [46] demonstrated that EA at Governing Vessel acupuncture points (GV6 and GV9) had a positive impact in a spinal cord injury model in rats. This effect is related to the neuroprotective effect of neurotrophin-3, which may increase the polarization of M2 microglia/macrophages. EA stimulation alleviates spinal nerve ligationeinduced neuropathic pain, at least in part through the inhibition of spinal glial activation
J.S. Vieira et al. [47]. EA also alleviates the muscle atrophy induced by sciatic nerve injection injury by upregulating agrin and epsilonacetylcholine receptors and downregulating gammaacetylcholine receptors [48]. Moreover, the use of lowfrequency transcutaneous electrical acupuncture point stimulation attenuates CCI-induced neuropathic pain, and its analgesic effect is likely mediated by the upregulation of opioid receptor expression in the DRG [49]. Taken together, these results indicate that there are specific peripheral (neural, skeletal muscle, connective tissue, and/or immunological) and central (opioid, cannabinoid and serotonin release, and receptor upregulation/ downregulation) effects of acupuncture that may be the key components to successful therapy and that to obtain such effects, the stimulation technique and the target tissue must be adapted to the condition treated. One limitation inherent in this study concerns the way the doses were determined. The functions of mediators are severalfold. Some of them activate neurons directly (e.g., the application of bradykinin, serotonin, or glutamate directly evokes action potentials), and/or they sensitize neurons for mechanical, thermal, and chemical stimuli (e.g., bradykinin and prostaglandins increase the excitability of neurons so that mechanical stimuli evoke action potentials at a lower threshold than under control conditions). In response to peripheral injury, many neuroactive mediators are released from blood cells, vascular endothelial cells, immune cells (including mast cells), and nerve fibers [50]. We selected the lowest dose of injection as 0.03 mg for all mediators and used 10- and 100-fold higher doses (0.3 mg and 3 mg). When the maximum dose did not change the withdrawal thresholds, the dose was increased to 30 mg (bradykinin and histamine). We suggest that these doses are related to the doses released when acupuncture is administered into the skin, but additional experimentation and/or binding studies are required to define the exact doses of injected mediators. In summary, our data are consistent with the concept that the neuroactive mediators released during acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia. These results may lead to clinical studies of pharmacopuncture application to acupuncture points for pain management. Additional experimentation is required to define the effects of neuroactive mediators on the afferent neurons in acupuncture-induced analgesia.
Financial support This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´gico (CNPq) Grant No. 447340/2014-0. J.S.V. received a Master’s Level Fellowship supported by the Coordenac ¸˜ ao de Aperfeic ¸oamento de Q16 Pessoal de Nı´vel Superior (CAPES).
Acknowledgments The authors are grateful for the excellent technical support from Luciana Costa Teodoro and Ze ´lia de Fa ´tima Fernandes.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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References
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[1] Patil S, Sen S, Bral M, Reddy S, Bradley KK, Cornett EM, et al. The role of acupuncture in pain management. Curr Pain Headache Rep 2016;20(4):22. [2] Wang G, Gao Q, Li J, Tian Y, Hou J. Impact of needle diameter on long-term dry needling treatment of chronic lumbar myofascial pain syndrome. Am J Phys Med Rehabil 2016;95(7): 483e94. [3] Ahn AC, Martinsen OG. Electrical characterization of acupuncture points: technical issues and challenges. J Altern Complement Med 2007;13(8):817e24. [4] Li AH, Zhang JM, Xie YK. Human acupuncture points mapped in rats are associated with excitable muscle/skin-nerve complexes with enriched nerve endings. Brain Res 2004;1012(1-2): 154e9. [5] Chen B, Li MY, Guo Y, Zhao X, Lim HC. Mast cell-derived exosomes at the stimulated acupoints activating the neuroimmuno regulation. Chin J Integr Med 2016. [6] 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(7):883e8. [7] Zhang D, Spielmann A, Wang L, Ding G, Huang F, Gu Q, et al. Mast-cell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2. Physiol Res 2012;61(1):113e24. [8] Chen W-H, Tzen JTC, Hsieh CL, Chen YH, Lin T-J, Chen S-Y, et al. Attenuation of TRPV1 and TRPV4 expression and function in mouse inflammatory pain models using electroacupuncture. Evid-Based Complement Altern Med: eCAM 2012;2012:636848. [9] Jiang Y-L, Yin X-H, Shen Y-F, He X-F, Fang J-Q. Low frequency electroacupuncture alleviated spinal nerve ligation induced mechanical allodynia by inhibiting TRPV1 upregulation in ipsilateral undamaged dorsal root ganglia in rats. Evid-Based Complement Altern Med: eCAM 2013;2013:170910. [10] Li WM, Cui KM, Li N, Gu QB, Schwarz W, Ding GH, et al. Analgesic effect of electroacupuncture on complete Freund’s adjuvant-induced inflammatory pain in mice: a model of antipain treatment by acupuncture in mice. Jpn J Physiol 2005;55(6):339e44. [11] Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33(1):87e107. [12] Yu Z, Luo L, Li Y, Wu Q, Deng S, Lian S, et al. Different manual manipulations and electrical parameters exert different therapeutic effects of acupuncture. J Tradit Chin Med 2014; 34(6):754e8. [13] Unger MD, Maus TP, Puffer RC, Newman LK, Currier BL, Beutler AS. Laminotomy for lumbar dorsal root ganglion access and injection in swine. J Vis Exp 2017;(128). [14] Sant’Anna MB, Kusuda R, Bozzo TA, Bassi GS, Alves-Filho JC, Cunha FQ, et al. Medial plantar nerve ligation as a novel model of neuropathic pain in mice: pharmacological and molecular characterization. Sci Rep 2016;6:26955. [15] Kim SK, Park JH, Bae SJ, Kim JH, Hwang BG, Min BI, et al. Effects of electroacupuncture on cold allodynia in a rat model of neuropathic pain: mediation by spinal adrenergic and serotonergic receptors. Exp Neurol 2005;195(2): 430e6. [16] Lu KW, Hsu CK, Hsieh CL, Yang J, Lin YW. Probing the effects and mechanisms of electroacupuncture at ipsilateral or contralateral ST36-ST37 acupoints on CFA-induced inflammatory pain. Sci Rep 2016;6:22123. [17] Baek YH, Huh JE, Lee JD, Choi DY, Park DS. Antinociceptive effect and the mechanism of bee venom acupuncture (Apipuncture) on inflammatory pain in the rat model of collagen-
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
induced arthritis: mediation by alpha2-Adrenoceptors. Brain Res 2006;1073e1074:305e10. Silva ML, Silva JR, Prado WA. The antinociceptive effect of electroacupuncture at different depths of acupoints and under the needling surface. Chin Med 2012;7:3. Chen HS, Qu F, He X, Liao D, Kang SM, Lu SJ. The antinociceptive effect and the possible mechanism of acupoint stimulation caused by chemical irritants in the bee venom pain model. Brain Res 2010;1355:61e9. Viguier F, Michot B, Hamon M, Bourgoin S. Multiple roles of serotonin in pain control mechanismseimplications of 5-HT(7) and other 5-HT receptor types. Eur J Pharmacol 2013;716(13):8e16. Sommer C. Serotonin in pain and analgesia. Mol Neurobiol 2004;30(2):117e25. Zhu X, Liu Z, Niu W, Wang Y, Zhang A, Qu H, et al. Effects of electroacupuncture at ST25 and BL25 in a Sennae-induced rat model of diarrhoea-predominant irritable bowel syndrome. Acupunct Med 2017;35(3):216e23. Zhu X, Liu Z, Qu H, Niu W, Gao L, Wang Y, et al. The effect and mechanism of electroacupuncture at LI11 and ST37 on constipation in a rat model. Acupunct Med 2016;34(3): 194e200. Kayser V, Latremoliere A, Hamon M, Bourgoin S. N-methyl-Daspartate receptor-mediated modulations of the antiallodynic effects of 5-HT1B/1D receptor stimulation in a rat model of trigeminal neuropathic pain. Eur J Pain 2011;15(5): 451e8. Chang FC, Tsai HY, Yu MC, Yi PL, Lin JG. The central serotonergic system mediates the analgesic effect of electroacupuncture on ZUSANLI (ST36) acupoints. J Biomed Sci 2004; 11(2):179e85. da Silva JR, da Silva ML, Prado WA. Electroacupuncture at 2/100 hz activates antinociceptive spinal mechanisms different from those activated by electroacupuncture at 2 and 100 hz in responder rats. Evid Based Complement Alternat Med 2013;2013:205316. Wang J, Liu J, Chen S, Gao Y, Meng F, Qiao L. Acupuncture effects on the hippocampal cholinergic system in a rat model of neuropathic pain. Neural Regen Res 2012;7(3):212e8. Silva JR, Silva ML, Prado WA. Analgesia induced by 2- or 100-Hz electroacupuncture in the rat tail-flick test depends on the activation of different descending pain inhibitory mechanisms. J Pain 2011;12(1):51e60. Ferreira SH, Nakamura M. II - Prostaglandin hyperalgesia: the peripheral analgesic activity of morphine, enkephalins and opioid antagonists. Prostaglandins 1979;18(2):191e200. Grando SA, Kist DA, Qi M, Dahl MV. Human keratinocytes synthesize, secrete, and degrade acetylcholine. J Invest Dermatol 1993;101(1):32e6. Carlton SM. Nociceptive primary afferents: they have a mind of their own. J Physiol 2014;592(16):3403e11. Honda K, Noma N, Shinoda M, Miyamoto M, Katagiri A, Kita D, et al. Involvement of peripheral ionotropic glutamate receptors in orofacial thermal hyperalgesia in rats. Mol Pain 2011;7:75. Farzin D, Asghari L, Nowrouzi M. Rodent antinociception following acute treatment with different histamine receptor agonists and antagonists. Pharmacol Biochem Behav 2002; 72(3):751e60. Mojtahedin A, Tamaddonfard E, Zanboori A. Antinociception induced by central administration of histamine in the formalin test in rats. Indian J Physiol Pharmacol 2008;52(3):249e54. Cannon KE, Chazot PL, Hann V, Shenton F, Hough LB, Rice FL. Immunohistochemical localization of histamine H3 receptors in rodent skin, dorsal root ganglia, superior cervical ganglia, and spinal cord: potential antinociceptive targets. Pain 2007; 129(1-2):76e92.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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10 [36] Poveda R, Fernandez-Duenas V, Fernandez A, Sanchez S, Puig MM, Planas E. Synergistic interaction between fentanyl and the histamine H3 receptor agonist R-(alpha)-methylhistamine, on the inhibition of nociception and plasma extravasation in mice. Eur J Pharmacol 2006;541(1-2):53e6. [37] Carlsson CP, Sundler F, Wallengren J. Cutaneous innervation before and after one treatment period of acupuncture. Br J Dermatol 2006;155(5):970e6. [38] Weidner C, Klede M, Rukwied R, Lischetzki G, Neisius U, Skov PS, et al. Acute effects of substance P and calcitonin gene-related peptide in human skinea microdialysis study. J Invest Dermatol 2000;115(6):1015e20. [39] Rosa AC, Fantozzi R. The role of histamine in neurogenic inflammation. Br J Pharmacol 2013;170(1):38e45. [40] Riedel W, Neeck G. Nociception, pain, and antinociception: current concepts. Z Rheumatol 2001;60(6):404e15. [41] Wu SY, Chen WH, Hsieh CL, Lin YW. Abundant expression and functional participation of TRPV1 at Zusanli acupoint (ST36) in mice: mechanosensitive TRPV1 as an “acupuncture-responding channel”. BMC Complement Altern Med 2014;14:96. [42] Lin JG, Hsieh CL, Lin YW. Analgesic effect of electroacupuncture in a mouse fibromyalgia model: roles of TRPV1, TRPV4, and pERK. PLoS One 2015;10(6), e0128037. [43] Zou W, Lin H, Liu W, Yang B, Wu L, Duan L, et al. Moxibustion relieves visceral hyperalgesia via inhibition of transient receptor potential vanilloid 1 (TRPV1) and heat shock protein (HSP) 70 expression in rat bone marrow cells. Acupunct Med 2016;34(2):114e9.
J.S. Vieira et al. [44] Zhang H, He S, Hu Y, Zheng H. Antagonism of cannabinoid receptor 1 attenuates the anti-inflammatory effects of electroacupuncture in a rodent model of migraine. Acupunct Med 2016;34(6):463e70. [45] Xu X, Wang MM, Sun ZL, Zhou DP, Wang L, Wang FQ, et al. Discovery of serum proteomic biomarkers for prediction of response to moxibustion treatment in rats with collageninduced arthritis: an exploratory analysis. Acupunct Med 2016;34(3):184e93. [46] Zhao J, Wang L, Li Y. Electroacupuncture alleviates the inflammatory response via effects on M1 and M2 macrophages after spinal cord injury. Acupunct Med 2017;35(3):224e30. [47] Liang Y, Qiu Y, Du J, Liu J, Fang J, Zhu J, et al. Inhibition of spinal microglia and astrocytes contributes to the antiallodynic effect of electroacupuncture in neuropathic pain induced by spinal nerve ligation. Acupunct Med 2016;34(1): 40e7. [48] Yu J, Wang M, Liu J, Zhang X, Yang S. Effect of electroacupuncture on the expression of agrin and acetylcholine receptor subtypes in rats with tibialis anterior muscular atrophy induced by sciatic nerve injection injury. Acupunct Med 2017; 35(4):268e75. [49] Yu X, Zhang F, Chen B. Effect of transcutaneous electrical acupuncture point stimulation at different frequencies in a rat model of neuropathic pain. Acupunct Med 2017;35(2):142e7. [50] Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 2009;139(2):267e84.
Please cite this article in press as: Vieira JS, et al., Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice, Journal of Acupuncture and Meridian Studies (2018), https://doi.org/ 10.1016/j.jams.2018.05.006
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