Probing the Effects and Mechanisms of Electroacupuncture at Ipsilateral or Contralateral ST36–ST37 Acupoints on CFA-induced Inflammatory Pain

Transient receptor potential vanilloid 1 (TRPV1) and associated signaling pathways have been reported to be increased in inflammatory pain signaling. There are accumulating evidences surrounding the therapeutic effect of electroacupuncture (EA). EA can reliably attenuate the increase of TRPV1 in mouse inflammatory pain models with unclear signaling mechanisms. Moreover, the difference in the clinical therapeutic effects between using the contralateral and ipsilateral acupoints has been rarely studied. We found that inflammatory pain, which was induced by injecting the complete Freund’s adjuvant (CFA), (2.14 ± 0.1, p < 0.05, n = 8) can be alleviated after EA treatment at either ipsilateral (3.91 ± 0.21, p < 0.05, n = 8) or contralateral acupoints (3.79 ± 0.25, p < 0.05, n = 8). EA may also reduce nociceptive Nav sodium currents in dorsal root ganglion (DRG) neurons. The expression of TRPV1 and associated signaling pathways notably increased after the CFA injection; this expression can be further attenuated significantly in EA treatment. TRPV1 and associated signaling pathways can be prevented in TRPV1 knockout mice, suggesting that TRPV1 knockout mice are resistant to inflammatory pain. Through this study, we have increased the understanding of the mechanism that both ipsilateral and contralateral EA might alter TRPV1 and associated signaling pathways to reduce inflammatory pain.

Inflammatory pain may result from thermal, chemical, or mechanical injuries that conduct signal transmission through nociceptors in the nervous system 1 . An inflammatory state can be initiated by injecting chemical agents such as the complete Freund's adjuvant (CFA) or carrageenan [2][3][4] . Inflammation could activate both mechanical and thermal transducers such as transient receptor potential vanilloid 1 (TRPV1) 5,6 , acid-sensing ion channels (ASICs) 7 , TRPV4 8 , TRPA1 9 , and TREK1/2 10 . Both local and systemic inflammation decrease pain threshold 11,12 . TRPV1 is a ion channel that is expressed in nociceptive neurons in the dorsal root ganglion (DRG), dorsal horn of the spinal cord (SCDH), the brain, and peripheral tissue 3,13 . TRPV1 is activated by acidic conditions, capsaicin, and heat (temperature > 43 °C) 13,14 , and activated TRPV1 can lead to mechanical or thermal hyperalgesia 15,16 . Alternatively, blocking TRPV1 can attenuate thermal hyperalgesia 17 . In a recent study, the authors found that TRPV1 played an important role in neuropathic pain 18 . Recent study found that thermal and mechanical hyperalgesia caused by CFA-induced inflammation led to increased TRPV1 expression in DRG neurons for 28 days 19 . Interestingly, the mechanism of TRPV1 is not as simple as described above. TRPV1 has been shown to respond to different stimuli depending on where the channel is located; peripherally expressed TRPV1 is involved in thermal hyperalgesia, whereas centrally expressed TRPV1 is involved in detecting both thermal and The picture shows that analgesic effect of EA could be detected immediately (30 min) after treatment. There was no statistically significant difference in following EA treatments at either right or left acupoints. (Control: saline injection; CFA: CFA injection; ipsilateral: EA treatment on the same side of CFA-induced inflammation; contralateral: EA treatment on the contralateral side of inflammation. *p < 0.05 as compared with control group, # p < 0.05 as compared with CFA group, n = 8. mechanical pain [20][21][22] . TRPV1 is highly expressed in the DRG 2 and also expressed in brain regions such as the cortex, cerebellum, hippocampus, and others area 23 . TRPV1 is also reported important in inflammatory pain and associated with pain-related molecules such as PKA 24 , PI3K 25 , and PKC 26 . Contralateral needling has been used to treat disease in traditional Chinese medicine for a long time. Koo et al. created an ankle sprain animal model, wherein the contralateral forelimbs could be treated using the endogenous opioid system 27 . A subsequent study indicated that the analgesic effect was mediated by the spinal α 2-adrenoceptor 28 . Somers et al. used contralateral transcutaneous electrical nerve stimulation (TENS) in rats with chronic constriction (CCI) and found improvements in mechanical, but not thermal, allodynia 29 . Yang et al. studied contralateral acupuncture in 2011, and they found that low frequency EA could reduce hyperalgesia induced by carrageenan and that the mechanism for the pain reduction used μ-opioid receptors in the SC 30 .
Acupuncture is a useful method for treating pain, and it has been used for a long time in Asia. Ancient books describe a variety of acupuncture techniques and different theories of pain management, including contralateral acupuncture. Due to its effectiveness in analgesia, people have investigated the mechanisms of pain relief acupuncture. In 1981, Melzack proposed the gate control theory to explain the analgesic effect of needling in trigger points 31 . Later the endorphin theory was proposed, which provided a stronger scientific evidence 32 . There are many studies about the analgesic mechanisms of acupuncture, which suggests that many factors are involved in the nociceptive pathway, including descending noradrenergic and serotonergic pathways 33,34 . In recent studies, it was found that EA might inhibit the release of proinflammatory cytokines such as IL-1, IL-6, TNF-α , and p38 35 . Recent article indicated that EA could reduce the expression of TRPV1 in a mice fibromyalgia pain model 36 .
Although theories have been proposed and studies have been conducted about contralateral acupuncture, they did not mention the difference between using ipsilateral and contralateral acupoints. The purpose of this study was to investigate whether there was a difference in analgesic effect following acupuncture at an ipsilateral acupoint compared to a contralateral acupoint. We did EA treatment at either the ipsilateral or contralateral side, and we found that they share similar mechanisms. We found that EA can reduce inflammatory pain induced by CFA and also alleviate nociceptive Nav sodium currents. EA also attenuated the overexpression of TRPV1 and associated signaling pathways that were increased after CFA injection. The potentiation of TRPV1 and associated pathways could be further avoided by deleting TRPV1 gene. Aforementioned mechanisms might be crucial and clear methods pertaining to EA analgesia. Electroacupuncture. There were four groups in this study: Control group; CFA group; ipsilateral group, EA was done at the same (right) side of CFA-induced inflammation, while the contralateral group was at left side. EA was done at ST36 and ST37 acupoints with stainless steel acupuncture needles (0.5 inch, 30 G, Yu-Kuang, Taiwan). We compared the therapeutic effect between ipsilateral and contralateral EA without sham control because it was ineffective 2,4,36 . The needles were inserted in 2-3 mm depth muscle layer at the acupoints. Electrical stimulation was produced by Trio 300 electrical stimulator (Grand Medical Instrument CO., LTD). The duration of each EA was 15 minutes; the setting of EA was 2 Hz in frequency, 1 mA in stimulation amplitude. EA was performed 30 min after CFA injection in day 1, in the morning (9:00 am-10:00 am) in day 2 and day 4. The procedure was done under anesthesia with 1% isoflurane in room temperature (25°C). The control group without EA treatment was also under anesthesia condition. Tissue sampling and Western blotting. We dissected out the dorsal aspect of the vertebral column using scissors and forceps. The spinal cord was further lifted out and dorsal horn was collected by scissors. L3-L5 DRG and SCDH neurons were immediately excised to extract proteins. Total proteins were prepared by homogenized DRG and SCDH in lysis buffer containing 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% NP− 40, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 0.02% NaN3 and 1 × protease inhibitor cocktail (AMRESCO). The extracted proteins (30 μg per sample assessed by BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat milk in TBS-T buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20), incubated with first antibody in TBS-T with 1% bovine serum albumin, and incubated for 1 hour at room temperature. Peroxidase-conjugated anti-rabbit antibody (1:5000) was used as a secondary antibody. The bands were visualized by an enhanced chemiluminescencent 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).

DRG primary cultures and Whole-cell patch-clamp recording.
Mice aged 8-12 weeks were killed by use of CO 2 to minimize their suffering. Lumbar (L3-L5) DRG neurons were dissected from ipsilateral site and placed in a tube containing DMEM and then transferred to DMEM with type I collagenase (0.125%, 120 min) for digestion at incubator at 37 °C. Neurons were then plated on poly-L-lysine-coated cover slides. All recordings were completed within 24 hours after plating. Glass pipettes (Warner Products 64-0792) were prepared (3-5 MΩ) with use of a vertical puller (NARISHIGE PC-10). Whole-cell recordings involved use of an Axopatch MultiClamp 700B (Axon Instruments). Stimuli were controlled and digital records captured with use of Signal 3.0 software and a CED1401 converter (Cambridge Electronic Design). Cells with a membrane potential more positive than − 40 mV were not accepted. The bridge was balanced in current clamping recording and series resistance was compensated 70% in voltage-clamping recording with Axopatch 700B compensation circuitry. Recording cells were superfused in artificial cerebrospinal fluid (ACSF) containing (in mM) 130 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 20 HEPES, adjusted to pH 7.4 with NaOH. ACSF solutions were applied by use of gravity. The recording electrodes were filled with (in mM) 100 KCl, 2 Na2-ATP, 0.3 Na3-GTP, 10 EGTA, 5 MgCl2, and 40 HEPES, adjusted to pH 7.4 with KOH. Osmolarity was approximately 300-310 mOsm. Capsaicin was prepared from a 100-μM stock solution (in 100% ethanol) to a final concentration of 1 μM in ACSF. All drugs were purchased from Sigma Chemical (St. Louis, MO, USA). (C) Expression of pCREB, Nav1.7, and Nav1.8. α -tubulin was used as the internal control. α -tubulin was used as the internal control. Con = Control; CFA = CFA induced inflammatory pain; Ipsi = electroacupuncture at ipsilateral site; Contra = electroacupuncture at contralateral site. * p < 0.05 compared to control group. # p < 0.05 compared to CFA group.
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 (p < 0.05 was considered statistically significant).

Results
Mechanical and thermal hyperalgesia induced by CFA injection was suppressed by both ipsilateral and contralateral EA treatments. To test whether ipsilateral or contralateral EA could equally reverse CFA-induced mechanical hyperalgesia, we compared responses to electric von Frey filaments at different days among control, CFA, ipsilateral, and contralateral EA groups. An injection of normal saline did not induce mechanical hyperalgesia (Fig. 1A, 4.15 ± 0.16 g, p < 0.05, compared with control group, n = 8), whereas an injection of CFA induced mechanical hyperalgesia in the hindpaw of mice on day 4 (Fig. 1A, 2.14 ± 0.1 g, p < 0.05, compared with control group, n = 8). The mechanical hyperalgesia was attenuated following either ipsilateral (Fig. 1A, 3.91 ± 0.21 g, p < 0.05, compared with CFA group, n = 8) or contralateral EA (Fig. 1A, 3.79 ± 0.25 g, p < 0.05, compared with CFA group, n = 8). Next, we utilized radial heat latency to define the degree of thermal hyperalgesia in mice. An injection of normal saline did not induce thermal hyperalgesia (Fig. 1B, 12.08 ± 0.79 s, p < 0.05, compared with control group, n = 8), whereas an injection of CFA induced thermal hyperalgesia on day 4 (Fig. 1B, 7.98 ± 0.25 s, p < 0.05, compared with control group, n = 8). However, the thermal hyperalgesia was abolished following either ipsilateral (Fig. 1B, 12.15 ± 0.83 s, p < 0.05, compared with CFA group, n = 8) or contralateral EA (Fig. 1B, 11.3 ± 0.68 s, p < 0.05, compared with CFA group, n = 8). These results demonstrated that EA at either the ipsilateral or the contralateral site of the ST36 acupoint could alleviate both mechanical and thermal hyperalgesia in CFA-induced inflammatory pain models.

Nav sodium currents were increased in CFA mice and attenuated by EA in DRG neurons.
To investigate whether the Nav sodium currents can be regulated by EA during CFA-induced inflammatory pain, we used whole-cell patch recordings to measure these currents. We depolarized DRG neurons from − 50 to + 30 mV to induce the currents. In the control group, the currents existed in DRG neurons and potentiated at 4 days after intraplantar CFA-induced inflammation (Fig. 2, p < 0.05, compared with control group, n = 8). Furthermore, EA significantly alleviated the increased Nav sodium currents, suggesting that the effect of the currents was reversible (Fig. 2, p < 0.05, compared with CFA group, n = 8).

Activation of TRPV1 with capsaicin dramatically increased the amplitude of Nav sodium currents.
To test whether TRPV1 activation can reliably enhance Nav sodium currents, we applied the TRPV1 agonist capsaicin directly to DRG neurons after the induction of Nav currents. We found that capsaicin increased   Our results show that TRPV1 acts as a receptor in inflammatory pain. Activation of TRPV1 increases the expression of pPKA, pPI3K, pPKC. Furthermore, pERK, pp38, pJNK, pAKT, and pCREB were also increased. Moreover, nociceptive Navs were increased for pain conduction. Aforementioned molecules could be attenuated in TRPV1 −/− mice. Nav currents in control DRG neurons (Fig. 3A, p < 0.05, compared with control group, n = 8). Next, we added capsaicin to inflamed DRG neurons, and we found that TRPV1 significantly potentiated the Nav currents (Fig. 3B, p < 0.05, compared with control group, n = 8). Furthermore, similar results were also obtained in DRG neurons from EA mice, suggesting its role in enhancing Nav currents (Fig. 3C, p < 0.05, compared with control group, n = 8). Together, these results indicated that activation of TRPV1 could enhance Nav currents in DRG neurons.

Discussion
Our results demonstrated that ipsilateral and contralateral acupuncture has similar analgesic effect in CFA-induced inflammatory pain. Electrophysiological results also indicated that Nav currents increased in CFA-induced inflammatory pain and further were attenuated by EA. Administration of TRPV1 agonist could significantly increase Nav sodium current. Western blotting analysis revealed that the expression levels of proteins Scientific RepoRts | 6:22123 | DOI: 10.1038/srep22123 in TRPV1 and associated signaling pathways were attenuated in the DRG of both EA treatment groups. Similarly, in the central SCDH, the expression of TRPV1 increased following CFA injections and significantly decreased following EA at either ipsilateral or contralateral sites. The data from western blotting experiments supported the data from behavioral experiments, which indicated that the efficacy of contralateral acupuncture was similar to that of ipsilateral acupuncture.
There were many theories about the mechanisms underlying the analgesic effect of EA or MA, including the gate control and the endogenous opiates theories. The mechanism underlying the analgesic effect of contralateral EA is not yet clear. A study indicates that spinal μ-opioid receptor plays a critical role in contralateral EA analgesic mechanism 30 . The authors performed EA at both ST36 and SP9 before carrageenan injection as a pretreatment. This design was not compatible with clinical use, but the researchers found a possible analgesic mechanism at the spinal level. Different acupoints are used to treat different diseases in the clinic, although some people consider that acupoints like LI4 and ST36 belong to "pain control. " In a study on contralateral acupoints analgesia, it was SI6 and not LI4 that provided pain relief in the contralateral hindpaw 28 . There is also a study that reported that many acupoints to have a similar analgesic effect in knee arthritis in mice 37 . Accordingly, descending regulation of opioid concept in peripheral level is not enough to completely explain the analgesic effect.
Recent study indicated that the amount of TRPV1 is higher in acupoints than it is in tissue along the meridian and nonacupoints 38 . They also concluded that the expression of TRPV1 was increased after EA stimulation. This is crucial for mediating the transduction of EA signals to the CNS 38 . From an anatomic point of view, an acupoint such as ST36 is TRPV1 rich, and this may be the reason why some acupoints are used for pain control 3,39 . Gao et al. reported that acupuncture at ST36 acupoint could significantly increase gastric motility amplitude in rats with atropine-induced gastric inhibition by 1-3 Hz frequency 40 . EA, but not sham control, can reliably control body weight by increasing TRPV1 in DRG and SC 41 . In this experiment, both ipsilateral and contralateral low frequency electric acupuncture had analgesic effects. Interestingly, there is a study about thermal and mechanical allodynia in a CCI (chronic construction incision) mouse model that used contralateral TENS instead of EA treatment. There was no statistical change in behavior following low frequency TENS (2 Hz) 29 . Only high frequency or high/low frequency TENS could alter neurotransmitters and improve mechanical pain. Low frequency stimulation can reduce cold allodynia in chronic nerve injury through spinal adrenergic and serotonergic receptors 37,42 . Therefore, EA and TENS may achieve analgesia in different conditions and using different pathways. Accordingly, we suggest that TRPV1 is abundant in the nerve endings of the DRG near ST36 acupoint that is responding for EA analgesia.
In a previous study, the expression of TRPV1 in DRG increased after inducing peripheral inflammation and decreased after EA application 2 . Coexpression of pPKCɛ and TPRV1 revealed in inflammatory state, which may be due to TPRV1 phosphorylation induced by activated pPKC 43 . TRPV1 and pPKCɛ are also reported for thermal pain 44 . Furthermore, PKC was coexpressed with TRPV1 in DRG under thermal stimuli 45 . There are fewer studies about pPKCɛ in the spinal level, but it is known that PKC may relieve inflammatory pain by modulating endogenous opioids such as the ouabain-like substance (OLS) via affecting c-Fos in the dorsal horn 46 . TRPV1 located in spinal cord should be essential in thermal and mechanical hyperalgesia 47 . Similar patterns were also obtained in pPKA and pPI3K, suggesting its crucial role in inflammatory pain.
The expression of pERK is also important for inflammation, it reacts rapidly in DRG and dorsal horn of spinal cord [48][49][50] . Researchers found that regulation of TRPV1 in DRG may be modulated by the Ras-MEK-ERK pathway in inflammation 51,52 . At the spinal level, ERK is active by inflammation and is essential for hyperalgesia 53 . EA can reduce inflammatory pain via modulating ERK, and the mechanism may be inhibiting COX2 and CREB-NK-1 in the dorsal horn 54 . Auricular electroacupuncture can reduce epilepsy by altering pPKC and pERK signaling pathways in kainic acid-treated rats 55 . Signaling pathways of pPKC and pERK were also reported to be involved in neuronal mechsanotransduction 56 . Nociceptive Nav1.7 and Nav1.8 sodium channels overexpression were also reduced by EA and genetic TRPV1 manipulation suggesting a crucial role in inflammatory pain. Our results showed reduction in pPKA, pPI3K, pPKC, and related molecules in the DRG and SC, and these results corresponded to the behavioral observations. The results of this experiment are compatible with previous studies. Both ipsilateral and contralateral EA can reduce nociceptive signaling in DRG and SCDH.

Conclusion
This study indicated a similar analgesic effect between ipsilateral and contralateral EA treatment on day 4 after inducing inflammatory pain in mice, and this effect was observed to become rapid after EA. Repeated EA treatment can further reduce pain but may reach a therapeutic plateau. Overexpression of TRPV1 and associated signaling pathways in both the DRG and SC were attenuated in TRPV1 −/− and EA-treated mice, and there was no significant difference between two EA treatments. This study provides a possible signaling mechanism of TRPV1 and relevant molecules (Fig. 8)