Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

Several voltage-gated sodium channels (Navs) from nociceptive nerve fibers have been identified as important effectors in pain signaling. The objective of this study is to investigate the electroacupuncture (EA) analgesia mechanism by changing the expression of Navs in mice dorsal root ganglia (DRG). We injected carrageenan and complete Freund's adjuvant (CFA) into the mice plantar surface of the hind paw to induce inflammation and examined the antinociception effect of EA at the Zusanli (ST36) acupoint at 2 Hz low frequency. Mechanical hyperalgesia was evaluated by using electronic von Frey filaments, and thermal hyperalgesia was assessed using Hargreaves' test. Furthermore, we observed the expression and quality of Navs in DRG neurons. Our results showed that EA reduced mechanical and thermal pain in inflammatory animal model. The expression of Nav1.7 and Nav1.8 was increased after 4 days of carrageenan- and CFA-elicited inflammatory pain and further attenuated by 2 Hz EA stimulation. The attenuation cannot be observed in Nav1.9 sodium channels. We demonstrated that EA at Zusanli (ST36) acupoint at 2 Hz low-frequency stimulation attenuated inflammatory pain accompanied by decreasing the expression of Nav1.7 and 1.8, rather than Nav1.9, sodium channels in peripheral DRG neurons.


Introduction
Recently, several studies have implied that voltage-gated sodium channels (Navs) might be involved in the development of hyperalgesia produced by inflammation [1][2][3]. Sensory neurons innervating the muscles are considered to sense muscle pain, and Navs are reported to participate in the process of inflammatory pain. Intraplantar injection of carrageenan and CFA is well documented to produce edema, as well as mechanical and thermal hyperalgesia, and has often been used as an inflammatory pain model [1][2][3][4]. Sodium channel-induced currents have been identified in CNS neurons [3] and in DRG (dorsal root ganglia) neurons [2] which significantly influence the threshold for action potential firing.
Acupuncture has been widely used for over 3000 years and has been based on the principles of traditional Chinese medicine. Acupuncture is known to stimulate the A -fibers [7] and modulate pain sensation by activating C-fibers through the meridian [8]. Acupuncture can be used therapeutically to treat diseases systematically [9]. The analgesic effect of acupuncture is already widely accepted. Several studies have suggested that acupuncture increases the release of endogenous opiates [10], serotonin [11], and adenosine to reduce pain [12]. Low-frequency electroacupuncture at 2 Hz induces enkephalins release to activate -receptor. In contrast, high-frequency stimulation releases dynorphins to activate -receptors [10].
Navs blockers are usually used for anesthesia and analgesia. To seek for more specific inhibitor with low side effects is possible. The rationale of this study is that Nav1.7, Nav1.8, and Nav1.9 are well known in inflammatory pain. The current study examines the crucial role of Navs and the effect of 2 Hz EA in mechanical and thermal hyperalgesia induced by carrageenan and CFA. We hypothesized that EA could alter expressions of Navs in both carrageenan-and CFA-induced inflammatory pain.

Animals and EA Pretreatment.
Adult ICR female mice aged 8 to 12 weeks were used in the experiment. The usage of these animals was approved by the Institute of Animal Care and Use Committee of China Medical University (Permit no. 101-116-N), Taiwan, following the Guide for the use of Laboratory Animals (National Academy Press). EA treatment was applied using stainless steel needles (12 mm, 32 G, Yu Kuang, Taiwan) which were inserted into the muscle layer to a depth of 2-3 mm at ST36 acupoint, which is therapeutic in both animal models and clinical study [11]. EA was administered immediately after the injection of carrageenan or CFA and performed every day at the same time (12:00-14:00). A Trio-300 (Japan) stimulator delivered electrical square pulses for 20 min with a 100 s duration and a 2 Hz frequency. The stimulation amplitude was 1 mA. The same treatment was given to nonacupoint (the upper lateral gluteal muscle but not GB30 acupoint) to be set as the sham control group entitled S-GM [13]. Another sham control group, entitled S-Acu, was induced by needling into ST36 acupoint without manipulation [14,15].

Inflammatory Pain Models.
Mice were anesthetized with 1-2% isoflurane and administered a single injection of 20 L saline (pH 7.4, buffered with 20 mM HEPES), CFA (0.5 mg/mL heat-killed M. tuberculosis Sigma, St. Louis, MO, MSA), or 3% carrageenan (lambda carrageenan and CFA, type IV; Sigma) in the plantar surface of the hind paw to induce intraplantar inflammation. Behavior tests were conducted at day 4 after induction of inflammation, and DRGs were harvested after behavior tests.

Animal Behavior of Mechanical and Thermal Hyperal-
gesia. Mechanical sensitivities were tested at 4 days after intraplantar injections. All experiments were performed at 30 min after EA (room temperature was approximately 25 ∘ C). Mechanical sensitivity was measured by testing the force of responses to stimulation with five applications of electronic von Frey filaments (North Coast Medical, Gilroy, CA, USA). Thermal pain was measured with five applications using Hargreaves' test IITC analgesiometer (IITC Life Sciences, Woodland Hills, CA, USA). Both hot-induced pain and coldinduced pain were measured using a hot/cold plate (IITC Life Sciences, Woodland Hills, CA, USA). Total of eight mice were used in each animal's behavior per group.

Immunoblotting
Assay. L3-L5 DRGs from 6 mice were immediately excised to extract proteins. Total proteins were prepared by homogenized DRG as previously described [13]. Peroxidase-conjugated anti-rabbit antibody (1 : 5000) was used as a secondary antibody. The bands were visualized by an enhanced chemiluminescent substrate kit (PIERCE) with LAS-3000 Fujifilm (Fuji Photo Film Co. Ltd). Where applicable, the image intensities of specific bands were quantified with NIH ImageJ software (Bethesda, MD, USA).

Statistical Analysis.
All statistic data are presented as the mean ± standard error. Statistical significance between control, inflammation, and EA group was tested using the ANOVA test, followed by a post hoc Tukey's test ( < 0.05 was considered statistically significant).

Immunohistochemistry Expression of Navs in DRG
Neurons. Our data showed that Nav1.7 sodium channels were distributed in L3-L5 DRG neurons (Figures 2(a) and  3(a)). Intraplantar injection of carrageenan or CFA reliably increased the expression of Nav1.7 sodium channels in L3-L5 DRG neurons (Figures 2(d) and 3(d)). Dramatically, Nav1.7 channels were negatively regulated to a normal level by applying 2 Hz EA treatment at ST36 acupoint (Figures 2(g) and 3(g)). EA-elicited downregulation of Nav 1.7 was not observed neither in sham-Acu (Figures 2(j) and 3(j)) nor in sham-GM groups (Figures 2(m) and 3(m)). Our results also show that Nav1.8 channels were expressed in DRG neurons in saline-injected neurons (Figures 2(b) and 3(b)). With the injection of carrageenan or CFA, Nav1.8 channels were greatly increased in DRG neurons (Figures 2(e) and 3(e)). The phenomenon was similar to previous results [2]. Importantly, 2 Hz EA at ST36 significantly reverses the overexpression of Nav1.8 channels in DRG neurons (Figures 2(h) and 3(h)). The effects were not obtained from sham-Acu (Figures 2(k) and 3(k)) and sham-GM groups (Figures 2(n) and 3(n)). The expression on Nav1.9 channels was observed in control group (Figures 2(c) and 3(c)). In carrageenan-and CFA-induced inflammation group, the expression of Nav1.9 was similar to that of the control one suggesting absence of the role of Nav1.9 in this model (Figures 2(f) and 3(f)). Similarly, the expression of Nav1.9 channels was not significantly different in the EAtreated group (Figures 2(i) and 3(i)), sham-Acu (Figures 2(l) and 3(l)), and sham-GM groups (Figures 2(o) and 3(o)).

Immunoblotting Quality of Navs in DRG Neurons.
We further showed that Nav1.7 and Nav1.8 channels were increased during carrageenan-and CFA-induced inflammatory pain in mice L3-L5 DRGs by using western blot technique (Figures 4(a) and 4(b)). In contrast, the expression of Nav1.9 sodium channels was not changed in this condition (Figure 4(c)). Our results suggested that Nav1.7 channels were attenuated by 2 Hz EA at ST36 acupoint in carrageenanand CFA-induced inflammation pain (Figure 4(a) displayed  Figure 4: Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan-and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered. (a) DRGs lysates were immunoreactive with specific antibodies to Nav1.7 and a substantially increased signal at the ipsilateral site, as compared to that of the saline-injected group. Nav1.7 protein levels were attenuated by EA at the ST36 acupoint, as compared to that of the carrageenan-and CFA-induced groups. (b) Nav1.8 displayed similar results to Nav1.7. The protein levels of S-Acu and S-GM were similar to inflamed but not EA group. (c) Nav1.9 protein levels were not changed in both the carrageenan-and CFA-injected sites. Nav1.9 protein levels were not attenuated by EA at the ST36 acupoint, as compared to those of the carrageenan-and CFA-induced groups, either. Nav1.9 proteins were not altered at the ipsilateral site of inflammation and EA stimulation.
a 46.2% decrease in the signal, as compared with the carrageenan group, = 6, < 0.05; a 78.8% decrease in the signal, as compared with the CFA group, = 6, < 0.05). Similar results were observed in Nav1.8 (Figure 4(b) displayed a 24.8% decrease in the signal, compared with the carrageenan group, = 6, < 0.05; a 30.7% decrease in the signal, compared with the CFA group, = 6, < 0.05). The protein levels of S-Acu and S-GM were similar to inflamed but not EA group suggesting acupoint specificity. Nav1.9 displayed no significant difference per group (Figure 4(c)). Accordingly, our results suggest that 2-Hz EA at the ST36 acupoint has the ability to ameliorate carrageenan-and CFA-induced overexpression of Nav1.7 and Nav1.8, rather than Nav1.9 sodium channels. All data were analyzed and presented in Figure 5.

Functional Analysis of TTX-R Currents Using Whole-Cell
Recording. To determine whether EA attenuates the neuronal excitability after CFA-induced inflammation pain model, we used whole-cell recording to record the TTX-R sodium currents in small-to-medium-size (<34 m) DRG neurons. In control group, TTX-R currents were obtained with membrane potential depolarized to −40 mV. However, intraplantar inflammation by CFA injection potentiated the amplitudes of TTX-R currents in DRG neurons. The potentiation of TTX-R currents was decreased in DRG neurons obtained from EAtreated group (Figure 6(a)). The relationship between membrane potential and Nav currents was plotted in Figure 6(b).

Discussion
In this study, we first established animal models of inflammatory pain by injection of carrageenan or CFA into hind paw. Animals with inflammatory pain showed mechanical and thermal hyperalgesia using a von Frey filament test, Hargreaves' test, and hot/cold plate tests. EA stimulation at the ST36 acupoint reduced inflammatory hyperalgesia in both carrageenan and CFA groups. Our results indicated that Nav1.7 and 1.8, but not Nav1.9, were upregulated in both carrageenan and CFA-induced hyperalgesia, which suggested the important role of Nav1.7 and 1.8 in inflammatory pain. We showed that EA at Zusanli (ST36) acupoint at 2 Hz lowfrequency stimulation reduced pain thresholds accompanied by decreasing the expression of Nav1.7 and 1.8, rather than Nav1.9, sodium channels in DRG neurons. Zhang et al. reported that EA at 10 Hz frequency significantly reduced CFA-induced hind paw edema. Moreover, EA attenuates inflammatory response through the hypothalamus-pituitary-adrenal axis (HPA) and the nervous system [14]. Recently, EA also suppresses the expression of neurokinin-1 in spinal cord dorsal horn induced by inflammation in rats [15]. These phenomena were not observed in sham control groups suggesting the acupoint-specific effect [14,15]. Our results were consistent with these studies that Evidence-Based Complementary and Alternative Medicine the antinociceptive effect was only observed in EA but not in sham-Acu and sham-GM groups. Numerous studies have investigated the role of different Navs in pain, neuron excitability, and action potential firing [4,17]. The most emphatic evidence implicating a specific ion channel participating in pain comes from studies on complete insensitivity to pain using gene knock mice [18,19]. Nav1.7 was greatly expressed in C-fiber free nerve endings, playing a crucial role in nociceptive information [20]. Recent studies have strongly supported Navs as potential analgesic drugs, according to antisense and knockout mice [6,21]. Derivatives from benzazepine and imidazopyridine were also developed to block Nav1.7 channels for pain treatment [22]. Our results clearly indicate that EA reliably attenuated carrageenanand CFA-induced inflammation pain by ameliorating Nav1.7 overexpression. This is the first paper regarding the functional role of acupuncture in pain manipulation and its novel findings pertaining to Nav1.7 channel alteration.
Chronic intrathecal Nav1.8 antisense injection successfully attenuated the Nav1.8-induced current and decreased mechanical allodynia after intraplantar CFA injection [23]. Developing a specific Navs channel blocker is possible for inflammatory pain. A-803467 is a novel specific blocker for the Nav1.8 channel and can ameliorate inflammatory pain in rats [24]. Nav1.9 is a TTX-R sodium channel greatly expressed in small diameter C-fibers and contributes to membrane properties, particularly in nociceptive neurons [25]. Nav1.9 is also suggested to regulate inflammatory pain thresholds [26]. Animal behavior studies have also demonstrated that deletion of Nav1.9 channel expression prevents inflammatory mediator-induced hyperalgesia [27,28]. Inflammatory mediators, such as PGE 2 , can reliably increase the Nav1.9 channel current in mice DRG neurons with G-protein activation [29]. Our data provide highly valuable results from investigating inflammation pain regarding ancient acupuncture mechanisms that can be further applied to clinical medicine.