THE ANATOMICAL RECORD 293:1207–1213 (2010)

Transcutaneous Electrical NerveStimulation on Yongquan Acupoint

Reduces CFA-Induced ThermalHyperalgesia of Rats via Down-Regulation

of ERK2 Phosphorylation andc-Fos Expression

LIN YANG, LIANXUE YANG, AND XIULAI GAO*

Department of Anatomy, Histology and Embryology, Capital Medical University,Beijing, People’s Republic of China

ABSTRACTActivation of extracellular signal-regulated kinase-1/2 (ERK1/2) and

its involvement in regulating gene expression in spinal dorsal horn, corti-cal and subcortical neurons by peripheral noxious stimulation contributeto pain hypersensitivity. Transcutaneous electrical nerve stimulation(TENS) is a treatment used in physiotherapy practice to promote analge-sia in acute and chronic inflammatory conditions. In this study, a totalnumber of 114 rats were used for three experiments. Effects of completeFreund’s adjuvant (CFA)-induced inflammatory pain hypersensitivity andTENS analgesia on ERK1/2 phosphorylation and c-Fos protein expressionwere examined by using behavioral test, Western blot, and immunostain-ing methods. We found that CFA injection caused an area of localizedswelling, erythema, hypersensitivity to thermal stimuli, the decreasedresponse time of hind paw licking (HPL), as well as upregulation of c-Fosprotein expression and ERK2 phosphorylation in the ipsilateral spinaldorsal horn and the contralateral primary somatosensory area of cortexand the amygdala of rats. TENS on Yongquan acupoint for 20 min pro-duced obvious analgesic effects as demonstrated with increased HPL tothermal stimuli of CFA-treated rats. In addition, TENS application sup-pressed the CFA-induced ERK2 activation and c-Fos protein expression.These results suggest that down-regulation of ERK2 phosphorylation andc-Fos expression were involved in TENS inhibition on CFA-induced ther-mal hyperalgesia of rats. Anat Rec, 293:1207–1213, 2010. VVC 2010 Wiley-Liss, Inc.

Keywords: CFA-induced inflammatory pain; TENS application;ERK1/2; c-Fos

Grant sponsor: National Natural Science Foundation of China;Grant number: 90209008; Grant sponsor: Beijing NaturalScience Foundation; Grant number: 5072008.

*Correspondence to: Xiulai Gao, MD, Department of Anatomy,Histology and Embryology, Capital Medical University, Beijing100069, China E-mail: yanglin@ccmu.edu.cn

Received 10 October 2009; Accepted 31 January 2010

DOI 10.1002/ar.21157Published online 13 April 2010 in Wiley InterScience (www.interscience.wiley.com).

VVC 2010 WILEY-LISS, INC.

Inflammatory and neuropathic pains are initiated bytissue damage/inflammation and nervous system lesions,and both are characterized by hypersensitivity at thesite of damage and in adjacent normal tissue (Woolf andSalter, 2000). Up to now, intraplantar or joint injectionof the complete Freund’s adjuvant (CFA) as an inflam-matory pain model has been widely used (Kim et al.,2006). The nociceptive system in the central nervous sys-tem (CNS) includes the spinal dorsal horn, the primarysomatosensory cortex (SI), and the amygdala (Woolf,2007). It has been reported that both inflammation andnerve injury induce transcriptional changes in dorsalhorn neurons, which are mediated by activation of themitogen-activated protein kinases (MAPK)-cAMP responseelement binding protein cascade (McCarson and Krause,1994; Hay et al., 1997). SI cortical neurons encode theintensity of tactile and nociceptive stimuli. Neurons inthe medial temporal lobe areas, including the amygdala,are involved in learning the association between aver-sive and neutral stimuli in normal conditions (Bucheland Dolan, 2000; Petrovic et al., 2000; Petrovic et al.,2002). The amygdala may contribute to pain processingboth directly by regulating nociceptive modulating sys-tems in the brainstem and indirectly by controlling be-havioral and autonomic output during pain (Petrovicet al., 2000; Petrovic et al., 2004).

c-Fos is the most extensively investigated member ofthe immediate early gene family (IEGs). It has been con-sidered a transcription factor and a cellular marker ofneural activity to identify activated neurons response toperipheral noxious stimulation in the CNS. Numerousstudies have demonstrated that noxious stimuli inducethe expression of c-Fos in the SI area, the amygdala,and the spinal dorsal horn (Kwon et al., 2004; Rohet al., 2006). The activation of ERK1/2, a well knownmember of MAPK family, is mediated via the conservedRas/Raf/MAPK pathway (Ji et al., 1999). Phosphoryl-ated ERK1/2 (p-ERK1/2) has been also used as amarker of neural activation (Ji et al., 2002; Kawasakiet al., 2004). However, few studies have been carriedout to investigate ERK1/2 activation and expression inthe SI area and the amygdala after inflammatory painstimulation.

Transcutaneous electrical nerve stimulation (TENS) isa treatment that has been shown to be effective for painrelief in a variety of conditions (Bjordal et al., 2003;Chao et al., 2007). Acupuncture is also an importanttherapeutic strategy in traditional Chinese medicine(Zhang et al., 2003; Kim et al., 2005). However, acupunc-ture typically involves penetration of the skin on thespecific points (named as acupoints) by a needle, andthe analgesic effect varies in the different conditions.TENS on acupoints may serve as a relatively safe andnoninvasive method to obtain the analgesic effect. Inthis study, we observed the TENS effects on phosphoryl-ation and protein expression levels of ERK1/2 and c-Fosexpression in the spinal dorsal horn, the SI area, andthe amygdala of rats following CFA injection usingTENS on Yongquan acupoint (KI 1). KI 1 is located onthe sole of the foot, at the indentation near the frontpart, between the second and third metatarsal bones,one-third of the distance from the webs of the toes tothe heel. KI 1 is usually applied to reduce pain on top ofthe head, blurry vision, throat numbness, and so forth(Lu and Liu, 1991).

MATERIALS AND METHODSAnimals and Drugs

All experiments were carried out on specific pathogen-free adult (range, 12–16 weeks of age) male Wistar rats(weighing range, 180–230 g). The animal protocols wereapproved by University Institutional Animal Care andUse Committee of Capital Medical University, and wereconsistent with the NIH policy on the use of experimen-tal animal (NIH Publications No. 80-23). A total of 114rats were used in six different groups (see Table 1).Group 1 (Saline only): Eighteen rats were injected with0.1 mL saline solution into the right ankle joint andtested or sacrificed 1 hr after injection. Out of 18 rats inGroup 1, six rats were used for hind paw licking (HPL)test, six for immunohistochemistry (IHC), and six forwestern blot (WB), respectively; Group 2 (Saline þ shamTENS): Six rats were injected with 0.1 mL saline solu-tion, treated with sham TENS for 20 min 0.5 hr afterinjection and used for HPL test at 1 hr after injection;Group 3 (Saline þ TENS): 18 rats were injected with 0.1mL saline solution, applied TENS for 20 min 0.5 hr afterinjection and tested or sacrificed at 1 hr after injection.Out of 18 rats in Group 3, six rats were used for HPLtest, six for IHC, and six for WB, respectively; Group 4(CFA only): 12 rats were used for HPL test and IHCrespectively, which were injected with 0.1 mL CFA andtested or sacrificed 1 hr after injection. Another 30 ratswere used for WB, which were sacrificed at five differenttime points (0.5, 1, 2, 24, and 48 hr after CFA injection);Group 5 (CFA þ sham TENS): Six rats were injectedwith 0.1 mL CFA, treated with sham TENS and used forHPL test at 1 hr after injection; Group 6 (CFA þ TENS):18 rats were injected with 0.1 mL CFA, applied TENSfor 20 min 0.5 hr after injection and tested or sacrificedat 1 hr after injection. Another six rats were treatedwith TENS 23.5 hr after CFA injection and sacrificed24 hr after CFA injection.

CFA (0.1mL per animal, Gibco, USA) was injected intothe right ankle joint to produce a pathological painmodel as previously reported (Abbadie et al., 1994).

TENS Application

A TENS device (HANS LH202H, Beijing Huawei Com-pany, Beijing, China) was used to induce antihyperalge-sia. The following parameters were used: low (LF, 2 Hz)and high (HF, 100 Hz) frequency alternately, with

TABLE 1. Experimental groups

HPL test IHC test WB test

1 Saline only 6 6 62 Saline þ Sham TENS 6 0 03 Saline þ TENS 6 6 64 CFA only 6 6 30a

5 CFA þ Sham TENS 6 0 06 CFA þ TENS 6 6 12b

Rats in different groups were sacrificed at 1 hr after salineor CFA injection.aRats were sacrificed at five different time points (0.5, 1, 2,24, and 48 hr after CFA injection).bRats were sacrificed at two different time points (1 and 24hr after CFA injection). TENS was applied for 20 min 0.5and 23.5 hr after CFA injection.

1208 YANG ET AL.

current intensity at range, 1–3 mA, and pulse durationat range, 0.2–0.6 ms. TENS was applied on KI 1 acu-point in the right hind limbs of rats at 0.5 hr or 23.5 hrafter the injection of CFA or physiological saline solutionand maintained for 20 min. Then the rats were testedfor the response time of HPL or were sacrificed at thetime point of 1 hr or 24 hr after CFA-injection. The pa-rameters of TENS regarding frequency, pulse duration,application time, and intensity were in accordance tothose used in routine physiotherapy practice (Han,2003). Rats with sham TENS treatment were placed in aTENS device without electric current.

Hyperalgesia was examined with measuring theresponse time of HPL to thermal stimuli using a hotplate analgesia meter (Hargreaves et al., 1988). The hotplate meter (YLS-6B, Huaibei Zhenghua Instruments &Equipments Company, China) was maintained at 52 �0.2�C. Because of individual variations, the animalsused in this study were chosen with the HPL responsetime shorter than 20 sec but longer than 6 sec under thenormal condition.

Immunostaining Study

Rats were deeply anesthetized with chloral hydrate(300 mg/kg, ip), transcardially perfused with 0.1 M phos-phate-buffered saline (PBS) followed by cold 4% parafor-maldehyde in PBS. The lumbar-sacral enlargement ofthe spinal cord and the brain were postfixed in 4% para-formaldehyde and dehydrated in 20% sucrose solutionovernight. Sections at 30 lm thickness were preparedfor c-Fos IHC staining. Sections were incubated with0.3% H2O2 in PBS (0.5 hr), blocked in 5% normal goatserum (60 min), and incubated overnight with primaryantibody c-Fos (1:100, Santa Cruz), then with biotinyl-ated goat anti-rabbit IgG. Finally, sections were devel-oped with DAB (Sigma, USA) staining. c-Fos positiveneurons in the spinal dorsal horn, the S1 area of the cor-tex, and the amygdala were counted in five representa-tive sections. Five areas (126.50 lm2 per area) werechosen for cell counts from each section.

Western Blotting

Rats were anesthetized with chloral hydrate (250 mg/kg, ip) and decapitated at each time point. The tissuesfrom the bilateral spinal cord, the SI area of the cortex,and the amygdala were immediately removed. The sec-tioned tissues were homogenized at 4�C in homogenizingbuffer and sonicated to dissolve the tissue completely aspreviously reported (Jiang et al., 2009). Then, theamounts of protein were determined by BCA kit (Pierce,USA).

The levels of ERK1/2 phosphorylation (p-ERK1/2) andprotein expression were determined using WB asdescribed previously (Zhang et al., 2007). Samples con-taining an equal amount of total protein were loaded on10% SDS-PAGE gel. After transferring to nitrocellulosemembranes (Schleicher & Schuell, USA), unspecificbinding was blocked by incubating in blocking buffer for1 hr. The blots were then incubated with primary anti-bodies overnight at 4�C. The following antibodies at a1:1,000 dilution were used: rabbit anti-mouse p-ERK1/2,rabbit anti-mouse total ERK1/2 (T-ERK1/2, Promega,USA), or mouse anti-b-actin (Sigma, USA). After incu-bating with an anti-rabbit or anti-mouse horseradishperoxidase-conjugated secondary antibody, protein wasvisualized using ECL-plus Kit (PerkinElmer Life Sci-ence, USA).

Data Analysis

Data were presented as mean � SD (standard devia-tion). Statistical analysis was conducted by one-wayanalysis of variance (ANOVA) followed by individualpost hoc multiple comparisons. A statistical differencewas accepted as significant at P < 0.05.

RESULTSEffects of CFA Injection and TENS Applicationon Thermal Hyperalgesia of Rats

CFA injection produced an area of localized swelling,erythema, and hypersensitivity to thermal stimuli,which persisted for the duration of experiment (48 hr).As shown in Table 2, the response time of HPL to ther-mal stimuli of CFA-injected rats at 1 hr decreased signif-icantly (P < 0.05, N ¼ 6) when compared with that ofcontrol group. However, TENS application could increasethe response time of HPL both in control and CFA injec-tion groups significantly (#P < 0.05, N ¼ 6).

Effects of CFA Injection and TENS Applicationon c-Fos Expression in the Spinal DorsalHorn, the SI Area of Cortex, and theAmygdala of Rats

As shown in Fig. 1A, c-Fos protein expressed in theipsilateral laminae I and II of the spinal dorsal horn,the contralateral SI area of cortex, and the amygdala.The numbers of c-Fos positive neurons in the ipsilat-eral spinal dorsal horn, the contralateral SI area ofcortex, and the amygdala increased significantly at 1hr post CFA injection (P < 0.05, N ¼ 6, Fig. 1B and E).In addition, 20 min TENS stimulation also increasedc-Fos protein expression significantly in the ipsilateralspinal dorsal horn, the contralateral SI, and the

TABLE 2. Changes in response time of hind paw licking of rats after CFAinjection and TENS application (N 5 6 per group)

Number (N)Before TENSstimulation (S)

After sham TENSstimulation (S)

After TENSstimulation (S)

Control 18 8.65 � 1.68 9.04 � 2.25 14.72 � 3.84#

CFA injection 18 5.12 � 1.29* 6.26 � 2.17* 9.86 � 1.58*,#

Versus control group, *P < 0.05; versus sham TENS stimulation, #P < 0.05; S, seconds.

TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION 1209

amygdala (Fig. 1C and E). However, 20 min TENSstimulation started at 0.5 hr post CFA injection couldreduce the number of c-Fos positive neurons in the ip-silateral spinal dorsal horn, but not in the contralat-eral SI area of cortex and the amygdala, whencompared with that of CFA group at 1 hr post CFAinjection (Fig. 1D and E).

Effects of CFA Injection and TENS Applicationon ERK1/2 Phosphorylation and ProteinExpression Levels in the Spinal Dorsal Horn,the SI Area of Cortex, and the Amygdala of Rats

The representative immunoblotting results of p-ERK1/2 and T-ERK1/2 were shown in Fig. 2A (ipsilateral

Fig. 1. Effects of CFA injection and TENS application on c-Fosexpressions in ipsilateral spinal dorsal horn, contralateral SI area ofcortex, and amygdala of rats. Typical results of c-Fos immunostainingin the ipsilateral laminae I and II of the spinal dorsal horn, contralateralSI area of cortex, and amygdala of rats following injections of saline

solution and sham transcutaneous electrical nerve stimulation (TENS,A), complete Freund’s adjuvant (CFA, B), TENS application (C), andTENS at 0.5 hr post CFA (D). *P < 0.05 versus control group, #P <0.05 versus CFA-treated group, N ¼ 6 per group.

1210 YANG ET AL.

spinal dorsal horn), B (contralateral SI area of cortex),and C (contralateral amygdala). The bands of p-ERK1/2,T-ERK1/2 as well as b-actin could be detected at 44 kDafor ERK1, 42 kDa for ERK2, and 43 kDa for b-actin,respectively. As shown in Fig. 3A, the phosphorylationlevels of ERK2, not ERK1, at 0.5 hr (118.9 � 5.2), 1 hr(115.2 � 5.4), 2 hr (128.6 � 8.4), 24 hr (123.1 � 4.6), and48 hr (125.1 � 3.6) post CFA injection in ipsilateral spi-nal dorsal horn increased significantly (P < 0.05) whencompared with that of control group (100%, N ¼ 6 pergroup). However, TENS application could inhibit theincrease of ERK2 phosphorylation in the ipsilateral spi-nal dorsal horn at 0.5 hr (105.7 � 4.6 vs. P-30 min, P <0.05) and 24 hr (108.5 � 4.2 vs. P-24 h, P < 0.05) postCFA injection.

Similarly, there were significant increase of ERK2phosphorylation both in contralateral SI (Fig. 2B and3B) and amygdala (Fig. 2C and 3C) at 0.5 hr, 1 hr, 2 hr,24 hr, and 48 hr post CFA injection. TENS applicationabolished the increase of ERK2 phosphorylation of CFA-injected rats (Fig. 2B, 2C, 3B and 3C). In addition, nochanges of total ERK1/2 protein expressions weredetected in the ipsilateral spinal dorsal horn, the contra-lateral SI area, and the amygdala of rats following CFAinjection (data not shown).

DISCUSSION

In this study, we undertook a detailed analysis on c-Fos protein expression and ERK1/2 phosphorylation lev-els in the spinal dorsal horn, the SI area of cortex, andthe amygdala of rats in response to CFA injection andTENS stimulation. Our results demonstrated that CFAinjection in ankle of rat in vivo causes significant c-Fosexpressions as well as ERK2 activation. The applicationof TENS stimulation on KI 1 could block the increases ofc-Fos expression and ERK2 phosphorylation as well asan obvious analgesia, which could be detected via theincreased response time of HPL in rats under thermalstimuli.

Ji et al. (Ji et al., 1999; 2002) found that CFA injectionof hind paw produced a persistent inflammation and a

Fig. 2. Typical Western blot results showed the changes in ERK1/2phosphorylation and protein expression in ipsilateral spinal dorsal horn(A), contralateral SI area of cortex (B), and amygdala (C) of rats follow-ing CFA and TENS. ERK1/2 and b-actin were detected at 42/44 and43 kDa, respectively. Control, saline solution, and sham TENS;P-0.5 hr, 0.5 hr post CFA; P-1 hr, 1 hr post CFA; P-2 hr, 2 hr postCFA; P-24 hr, 24 hr post CFA; P-48 hr, 48 hr post CFA; P-0.5 hr þTENS, 20 min TENS at 0.5 hr post CFA; P-24 hr þ TENS, 20 minTENS at 24 hr post CFA.

Fig. 3. Quantitative analysis of ERK1/2 phosphorylation level in ipsi-lateral spinal dorsal horn (A), and contralateral SI area of cortex (B),and amygdala (C) of rats following CFA and TENS. Fifty-four rats wererandomly divided into the following nine groups: control (N ¼ 6), P-0.5hr (N ¼ 6), P-1 hr (N ¼ 6), P-2 hr (N ¼ 6), P-24 hr (N ¼ 6), P-48 hr(N ¼ 6), P-0.5 hr þ TENS (N ¼ 6) and P-24 hr þ TENS (N ¼ 6). Theresults showed that TENS application suppressed CFA-induced ERK2activation in the ipsilateral spinal cord, contralateral SI area of cortex,and amygdala of rats. *P < 0.05 versus control.

TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION 1211

sustained ERK activation in neurons of the superficialdorsal horn. Zhuang et al. (Zhuang et al., 2005) reportedthat fifth lumbar spinal nerve ligation induced an imme-diate (<10 min) but transient (<6 hr) induction of ERKphosphorylation restricted to neurons in the superficialdorsal horn. These findings suggested that the involve-ment of p-ERK in peripheral inflammatory pain hyper-sensitivity may be contributed to the regulation of targetgenes such as IEGs. In this study, we found that CFAinduced a persistent ERK2 activation (>48 hr) not onlyin the spinal cord but also in the SI area of cortex andthe amygdala.

Our data also provided evidence that CFA injectioncauses an obvious increased expression of c-Fos in thespinal cord, the SI area of cortex, and the amygdala ofrats. c-Fos is a very important IEGs member, which hasbeen shown to take part in injury-related cellular mech-anisms in many different systems including neuronaltransport blockade (Guo et al., 2004; Wang et al., 2006).c-Fos induction by CFA had already been demonstratedin neurons of the spinal cord and cortex (Abbadie et al.,1994; Bellavance and Beitz, 1996; Cruz et al., 2007).Furthermore, we found that CFA injection induced c-Fosexpressions in the amygdala. Amygdala is a complexsubcortex structure, which mediates a specific aspect ofemotional behaviors including the process of pain encod-ing and modulation. Studies have demonstrated that theamygdala has abundant opiates receptors and partici-pates in both opioid analgesia and acupuncture analge-sia (Fields, 2000; Napadow et al., 2007). The increases ofc-Fos expression and ERK2 phosphorylation in theamygdala suggested that it may integrate the nocicep-tive information from the spinal cord and other corticalareas and then generate the emotional and behavioralresponses on pain.

Another important finding of this study is that TENSapplication on KI 1 could inhibit CFA-induced ERK2activation and c-Fos expression. c-Fos and ERK expres-sions were reduced by ERK inhibitor in CFA-treatedrats implicated the important role for ERK/c-Fos-de-pendent transduction pathways in acute and chronicmodulation of nociceptive stimulation (Giles et al.,2007). In addition, TENS on acupoint induced anobvious analgesic effect by the increased HPL of CFA-treated rats. TENS has been used to promote analgesiafor nearly 40 years. However, the use of TENS combinedwith acupoint has not been widespread despite of itsprominent improvement in function of the affectedregion especially at pain relieving. Studies supported thehypothesis that therapeutic acupuncture was mediatedby opioidergic and/or monoamingergic neurotransmis-sion involving the brain stem, the thalamus, and theamygdala action (Garrison and Foreman, 1994; Dhondet al., 2007). Afferent spinal gating and the diffuse nox-ious inhibitory control may be involved in short-term an-algesic effects of TENS stimulation (Carlsson, 2002).Our primary fMRI study demonstrated that the brainnetwork associated with the amygdala implicated inboth pain sensation and pain modulation, and acupunc-ture might change this amygdala-specific brain networkinto a functional state to affect pain perception and painmodulation (Qin et al., 2008).

In conclusion, these findings suggest that down-regu-lation of ERK2 phosphorylation and c-Fos expression inthe spinal dorsal horn as well as in the SI area of cortex

and the amygdala were involved in TENS inhibition onCFA-induced thermal hyperalgesia of rats. It also pro-vide a reasonable explanation for the actual analgesiceffect of acupuncture as well as direct evidence againstthat an acupuncture point may have its own functionalspecificity. However, further experiment on the mecha-nism of TENS-induced dephosphorylation of ERK afterCFA stimulation should be carried out.

ACKNOWLEDGMENTS

The authors thank Dr. Shewei Guo, Dr. Hao Wang,Dr. Chengji Liu, and Dr. Xiangning Bu for technical as-sistance. They are grateful to Professors Junfa Li, MingZhang, Deshan Zhou, and Weiming Duan for readingand commenting earlier drafts of this article. Authorsalso thank all participants for their time and patience.

LITERATURE CITED

Abbadie C, Besson JM, Calvino B. 1994. C-Fos expression in thespinal-cord and pain-related symptoms induced by chronic arthri-tis in the rat are prevented by pretreatment with freund adju-vant. J Neurosci 14:5865–5871.

Bellavance LL, Beitz AJ. 1996. Altered c-fos expression in the para-brachial nucleus in a rodent model of CFA-induced peripheralinflammation. J Comp Neurol 366:431–447.

Bjordal JM, Johnson MI, Ljunggreen AE. 2003. Transcutaneouselectrical nerve stimulation (TENS) can reduce postoperativeanalgesic consumption. A meta-analysis with assessment of opti-mal treatment parameters for postoperative pain. Eur J Pain7:181–188.

Buchel C, Dolan RJ. 2000. Classical fear conditioning in functionalneuroimaging. Curr Opin Neurobiol 10:219–223.

Carlsson C. 2002. Acupuncture mechanisms for clinically relevantlong-term effects—reconsideration and a hypothesis. AcupunctMed 20:82–99.

Chao AS, Chao A, Wang TH, Chang YC, Peng HH, Chang SD, ChaoA, Chang CJ, Lai CH, Wong AM. 2007. Pain relief by applyingtranscutaneous electrical nerve stimulation (TENS) on acupunc-ture points during the first stage of labor: a randomized double-blind placebo-controlled trial. Pain 127:214–220.

Cruz CD, Ferreira D, McMahon SB, Cruz F. 2007. The activation ofthe ERK pathway contributes to the spinal c-fos expressionobserved after noxious bladder stimultion. Somatoens Mot Res24:15–20.

Dhond RP, Kettner N, Napadow V. 2007. Do the neural correlates ofacupuncture and placebo effects differ? Pain 128:8–12.

Fields HL. 2000. Pain modulation: expectation, opioid analgesia andvirtual pain. Prog Brain Res 122:245–253.

Garrison DW, Foreman RD. 1994. Decreased activity of spontaneousand noxiously evoked dorsal horn cells during transcutaneouselectrical nerve stimulation (TENS). Pain 58:309–315.

Giles PA, Trezise DJ, King AE. 2007. Differential activation of pro-tein kinases in the dorsal horn in vitro of normal and inflamedrats by group I metabotropic glutamate receptor subtypes. Neuro-pharmacology 53:58–70.

Guo ZL, Moazzami AR, Longhurst JC. 2004. Electroacupunctureinduces c-Fos expression in the rostral ventrolateral medulla andperiaqueductal gray in cats: relation to opioid containing neurons.Brain Res 1030:103–115.

Han JS. 2003. Acupuncture: neuropeptide release produced by elec-trical stimulation of different frequencies. Trends Neurosci 26:17–22.

Hargreaves K, Dubner R, Brown F, Flores C, Joris J. 1988. A newand sensitive method for measuring thermal nociception in cuta-neous hyperalgesia. Pain 32:77–88.

Hay CH, Trevethick MA, Wheeldon A, Bowers JS, de Belleroche JS.1997. The potential role of spinal cord cyclooxygenase-2 in the

1212 YANG ET AL.

development of Freund’s complete adjuvant-induced changes inhyperalgesia and allodynia. Neuroscience 78:843–850.

Ji RR, Baba H, Brenner GJ, Woolf CJ. 1999. Nociceptive-specificactivation of ERK in spinal neurons contributes to pain hypersen-sitivity. Nat Neurosci 2:1114–1119.

Ji RR, Befort K, Brenner GJ, Woolf CJ. 2002. ERK MAP kinaseactivation in superficial spinal cord neurons induces prodynorphinand NK-1 upregulation and contributes to persistent inflamma-tory pain hypersensitivity. J Neurosci 22:478–485.

Jiang J, Yang W, Huang P, Bu X, Zhang N, Li J. 2009. Increasedphosphorylation of Ets-like transcription factor-1 in neurons ofhypoxic preconditioned mice. Neurochem Res 34:1443–1450.

Kawasaki Y, Kohno T, Zhuang ZY, Brenner GJ, Wang H, Van DerMC, Befort K, Woolf CJ, Ji RR. 2004. Ionotropic and metabotropicreceptors, protein kinase A, protein kinase C, and Src contributeto C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leadingto central sensitization. J Neurosci 24:8310–8321.

Kim JH, Chung JY, Kwon YK, Kim KJ, Yang CH, Hahm DH, LeeHJ, Pyun KH, Shim I. 2005. Acupuncture reduces alcohol with-drawal syndrome and c-Fos expression in rat brain. Am J ChinMed 33:887–896.

Kim JH, Kim HK, Park YI, Sohn IC, Choi DO, Kim MS, Park BR.2006. Moxibustion at ST36 alleviates pain in complete Freund’sadjuvant-induced arthritic rats. Am J Chin Med 34:57–67.

Kwon YB, Han HJ, Beitz AJ, Lee JH. 2004. Bee venom acupointstimulation increases Fos expression in catecholaminergic neu-rons in the rat brain. Mol Cells 17:329–333.

Lu R, Liu M. 1991. Clinical application of single acupoint for treat-ment. J Tradit Chin Med 11:284–285.

McCarson KE, Krause JE. 1994. NK-1 and NK-3 type tachykininreceptor mRNA expression in the rat spinal cord dorsal horn isincreased during adjuvant or formalin-induced nociception.J Neurosci 14:712–720.

Napadow V, Kettner N, Liu J, Li M, Kwong KK, Vangel M, MakrisN, Audette J, Hui KK. 2007. Hypothalamus and amygdalaresponse to acupuncture stimuli in Carpal Tunnel Syndrome.Pain 130:254–266.

Petrovic P, Carlsson K, Petersson KM, Hansson P, Ingvar M. 2004.Context-dependent deactivation of the amygdala during pain.J Cogn Neurosci 16:1289–1301.

Petrovic P, Petersson KM, Ghatan PH, Stone-Elander S, Ingvar M.2000. Pain-related cerebral activation is altered by a distractingcognitive task. Pain 85:19–30.

Petrovic P, Petersson KM, Hansson P, Ingvar M. 2002. A regressionanalysis study of the primary somatosensory cortex duringpain10. Neuroimage 16:1142–1150.

Qin W, Tian J, Bai L, Pan X, Yang L, Chen P, Dai J, Ai L, Zhao B,Gong Q, Wang W, von Deneen KM, Liu Y. 2008. FMRI connectiv-ity analysis of acupuncture effects on an amygdala-associatedbrain network. Mol Pain 4:1–17.

Roh DH, Kim HW, Yoon SY, Kang SY, Kwon YB, Cho KH, Han HJ,Ryu YH, Choi SM, Lee HJ, Beitz AJ, Lee JH. 2006. Bee venominjection significantly reduces nociceptive behavior in themouse formalin test via capsaicin-insensitive afferents. J Pain7:500–512.

Wang TT, Yuan WL, Ke Q, Song XB, Zhou X, Kang Y, Zhang HT,Lin Y, Hu YL, Feng ZT, Wu LL, Zhou XF. 2006. Effects of electro-acupuncture on the expression of c-jun and c-fos in spareddorsal root ganglion and associated spinal laminae following re-moval of adjacent dorsal root ganglia in cats. Neuroscience140:1169–1176.

Woolf CJ. 2007. Central sensitization: uncovering the relationbetween pain and plasticity. Anesthesiology 106:864–867.

Woolf CJ, Salter MW. 2000. Neuronal plasticity: increasing the gainin pain. Science 288:1765–1769.

Zhang N, Gao G, Bu X, Han S, Fang L, Li J. 2007. Neuron-specificphosphorylation of c-Jun N-terminal kinase increased in the brainof hypoxic preconditioned mice. Neurosci Lett 423:219–224.

Zhang YQ, Ji GC, Wu GC, Zhao ZQ. 2003. Kynurenic acid enhanceselectroacupuncture analgesia in normal and carrageenan-injectedrats. Brain Res 966:300–307.

Zhuang ZY, Gerner P, Woolf CJ, Ji RR. 2005. ERK is sequentiallyactivated in neurons, microglia, and astrocytes by spinal nerve li-gation and contributes to mechanical allodynia in this neuro-pathic pain model. Pain 114:149–159.

TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION 1213