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The Journal of Pain, Vol 7, No 7 (July), 2006: pp 500-512Available online at www.sciencedirect.com

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ee Venom Injection Significantly Reduces Nociceptive Behaviorn the Mouse Formalin Test via Capsaicin-Insensitive Afferents

ae-Hyun Roh,* Hyun-Woo Kim,* Seo-Yeon Yoon,* Seuk-Yun Kang,*oung-Bae Kwon,† Kwang-Hyun Cho,‡ Ho-Jae Han,§ Yeon-Hee Ryu,� Sun-Mi Choi,�

ye-Jung Lee,¶ Alvin J. Beitz,# and Jang-Hern Lee*Department of Veterinary Physiology, College of Veterinary Medicine and School of Agricultural Biotechnology,eoul National University, Seoul, South Korea.Department of Pharmacology, andDepartment of Psychiatry, Institute for Medical Science, Chonbuk National University Medical School, Jeonju,outh Korea.Hormone Research Center and College of Veterinary Medicine, Chonnam National University, Gwangju, Southorea.

Department of Medical Research, Korea Institute of Oriental Medicine, Daejeon, South Korea.Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyunghee University, Seoul, Southorea.Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Staul, Minnesota.

Abstract: Peripheral bee venom (BV) administration produces 2 contrasting effects, nociception andantinociception. This study was designed to evaluate whether the initial nociceptive effect induced byBV injection into the Zusanli acupoint is involved in producing the more prolonged antinociceptiveeffect observed in the mouse formalin test, and whether capsaicin-sensitive primary afferents areinvolved in these effects. BV injection into the Zusanli point increased spinal Fos expression but notspontaneous nociceptive behavior. BV pretreatment 10 minutes before intraplantar formalin injectiondose-dependently attenuated nociceptive behavior associated with the second phase of the formalintest. The destruction of capsaicin-sensitive primary afferents by resiniferatoxin (RTX) pretreatmentselectively decreased BV-induced spinal Fos expression but did not affect BV-induced antinociception.Furthermore, BV injection increased Fos expression in tyrosine hydroxylase immunoreactive neuronsin the locus caeruleus, and this expression was unaltered by RTX pretreatment. Finally, BV’s antino-ciception was blocked by intrathecal injection of 10 �g idazoxan, and this effect was not modified byRTX pretreatment. These findings suggest that subcutaneous BV stimulation of the Zusanli pointactivates central catecholaminergic neurons via capsaicin-insensitive afferent fibers without induc-tion of nociceptive behavior. This in turn leads to the activation of spinal �2-adrenoceptors, whichultimately reduces formalin-evoked nociceptive behaviors.Perspective: This study demonstrates that BV acupuncture produces a significant antinociception with-out nociceptive behavior in rodents, which is mediated by capsaicin-insensitive afferents and involvesactivation of central adrenergic circuits. These results further suggest that BV stimulation into this acu-puncture point might be a valuable alternative to traditional electrical or mechanical acupoint stimulation.

© 2006 by the American Pain Society
Key words: Bee venom, capsaicin-sensitive primary afferents, formalin test, fos, resiniferatoxin.
eceived May 10, 2005; Revised February 3, 2006; Accepted February 4,006.upported by a grant (M103KV010009 03K2201 00940) from the Brainesearch Center of the 21st Century Frontier Research Program funded byhe Ministry of Science and Technology of the Republic of Korea and byRC program of KOSEF (R11-2005-014) as well as the Brain Korea 21roject.

Address requests for reprints to Jang-Hern Lee, DVM, PhD, Department ofVeterinary Physiology, College of Veterinary Medicine, Seoul NationalUniversity, Seoul 151-742, South Korea. E-mail: [email protected]/$32.00© 2006 by the American Pain Societydoi:10.1016/j.jpain.2006.02.002

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501ORIGINAL REPORT/Roh et al

ee venom (BV) injection can produce both an initialnociceptive effect and a prolonged antinociceptiveeffect. BV contains a number of potential pain-pro-

ucing substances including melittin, histamine, andhospholipase A2, and therefore it is not surprising thateveral reports described a nociceptive effect after in-raplantar injection.3,18,20 Luo et al23 have also reportedhat intraplantar BV injection significantly increases Fosxpression in the spinal cord dorsal horn of anesthetizedats. In contrast, subcutaneous injection of diluted BVnto an acupoint, termed apipuncture, has been usedlinically in Oriental medicine to produce a potent anal-esic effect. In support of this alternative medicine ap-roach recent experimental studies in our laboratoriesave demonstrated that subcutaneous injection of BV0.01 to 1 mg/kg) into the Zusanli acupuncture point pro-uces prominent antinociceptive and antihyperalgesicffects in animal models of acute and persistent pain,espectively.15,17,18,30 The above studies indicate that aichotomy exists with respect to the physiologic re-ponse to subcutaneous injection of BV. On one hand,ntraplantar injection of BV and its major constituent,

elittin, produces robust nociceptive behaviors and hy-ersensitivity in rodents, whereas BV injection into theusanli acupoint, on the other hand, produces little no-iceptive behaviors, but rather a significant antinocicep-ive effect in a variety of animal pain models. Despite thepparent conflicting data in the literature regarding theonsequences of BV injection, there have been no studieshat have examined a possible relationship between BV’sociceptive and antinociceptive effects, particularly withespect to BV injection into an acupoint. To begin toxamine this issue, we evaluated whether the intensityf the BV-induced nociception (as measured by sponta-eous pain behavior) and BV-induced neuronal activa-ion (as measured by spinal Fos expression) produced bynjection into the Zusanli acupoint is correlated with thentensity of the BV-induced antinociception (BVAN) inhe mouse formalin test. Because both BV’s nociceptivend antinociceptive effects appear to involve activationf primary afferent fibers, we also explored whether pri-ary afferent axons expressing the vanilloid receptor 1

TRPV1) were involved in either of these effects.TRPV1-expressing primary afferent neurons, termed

apsaicin-sensitive primary afferents (CSPAs), have beenecognized as nociceptive polymodal C-fibers whose cellodies are located in dorsal root ganglia. Functionally,SPAs are known to play a major role in nociceptiveransmission.2,37 Recent studies with a BV-induced painodel have shown that CSPAs play a critical role in me-iating both the thermal and mechanical hyperalgesia

nduced by BV injection.4 On the other hand, capsaicin-nduced excitation of TRPV1 receptors has also beenhown to be involved in counter-irritation mechanismsie, pain in one part of the body can be used to controlain in another part) that are involved in inhibiting theevelopment of subsequent nociceptive behaviors and

nflammatory reactions at distant body sites in theat.1,33 On the basis of these studies, we hypothesized
hat BV activation of CSPAs not only elicits a nociceptive t
ignal, but that activation of CSPAs can simultaneouslyroduce BVAN via a counter-irritation mechanism that

nvolves activation of the descending pain inhibitory sys-em (DPIS). To test this hypothesis we examined whetherhe depletion of CSPAs by using resiniferatoxin (RTX)retreatment35 could modify BV-induced spinal Fos ex-ression and BVAN in the formalin test.BVAN involves activation of primary afferent fibers asiscussed above. We have previously reported that BVAN

s blocked by intrathecal pretreatment with the �2-adre-oceptor antagonists idazoxan or yohimbine in severalifferent pain models.16,17,30 This implies that BVAN isediated by the activation of spinal �2-adrenoceptors,hich are known to be involved with the DPIS.24 In this

egard, we have recently shown that peripheral BV injec-ion effectively increases brainstem catecholaminergiceuronal activity including the activity of the locus caer-leus (LC).19 Therefore, the final objective of this studyas to evaluate whether RTX pretreatment also affectsV-induced catecholaminergic neuronal activity in theC and subsequent spinal �2-adrenoceptor activation.

aterials and Methods

nimalsExperiments were performed on male ICR mice weigh-

ng 20 to 25 g. All experimental animals were obtainedrom the Laboratory Animal Center of Seoul Nationalniversity. They were housed in colony cages with freeccess to food and water and maintained in tempera-ure- and light-controlled rooms (23°C � 2°C, 12/12-houright/dark cycle with lights on at 7:00 AM) for at least 1eek before the study. All of the methods used in theresent study were approved by the Animal Care and Useommittee at Seoul National University and conform toational Institutes of Health guidelines (NIH publicationo. 86-23, revised 1985). In addition, the ethical guide-

ines for investigating experimental pain in conscious an-mals recommended by the International Association forhe Study of Pain were followed.43

V Administration and RTX PretreatmentTo evaluate the effect of BV injection into the Zusanli

cupoint on spinal Fos expression and nociceptive behav-ors as well as on BVAN in the formalin test in naive mice,V from Apis mellifera (Sigma, St Louis, Mo) was dis-olved in physiologic saline (20 �L) at doses ranging from.001 to 10 mg/kg. A therapeutic dose of 0.005 to 0.5g/kg of BV is typically used to produce analgesia in

uman patients and is considered to be safe because thisose range does not appear to affect the central ner-ous, cardiovascular, respiratory, and gastrointestinalystems.14 Accordingly, the dose range of BV used in theresent study encompassed these clinically used doses.iluted BV was subcutaneously administered into theusanli acupoint of the right hind limb located on theateral side of the stifle joint adjacent to the anteriorubercle of the tibia as previously described.16 Animals in
he control group received an injection of vehicle into

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502 Mechanism of Bee Venom–Induced Antinociception

he Zusanli acupoint. Five mice per individual groupere used for analysis of the effects of different doses ofV on spinal cord Fos expression and on BV-induced no-iceptive behavior. Eight or more mice were included inach BV treatment or control group for behavioral anal-sis in the formalin test.To evaluate the potential role of CSPAs in BV-induced

pinal Fos expression and on BVAN in the formalin test,nly the high (10 mg/kg), middle (0.1 mg/kg), and low0.001 mg/kg) doses of BV were used in these experi-ents. The extremely potent capsaicin analog RTX (0.2g/kg; Sigma) was dissolved in a mixture of 10% Tween

0, 10% ethanol, and 80% normal saline.12,27 Either RTXr vehicle (10% Tween 80, 10% ethanol, and 80% nor-al saline; SHAM) was injected subcutaneously in a vol-me of 50 �L into the scruff of the neck of the mousenesthetized with 3% isoflurane in a mixture of N2O/O2

as 2 weeks before performing BV-induced Fos immuno-istochemistry and the formalin test. We waited 2 weeksfter RTX pretreatment to test the possible role of CSPAs,hich is based on the timeframe used in a previous

tudy.32 To confirm that RTX treatment destroyed CSPAs,diluted capsaicin solution (0.01%, dissolved in saline)as dropped into cornea, and then the number of eyeipes was counted for 1 minute on the day before BV-

nduced Fos immunohistochemistry and formalin injec-ion (SHAM, n � 24; RTX, n � 29). In addition to countingapsaicin-induced eye wipes, TRPV1 immunohistochem-stry was performed on both the dorsal root ganglionDRG) and the spinal cord at the completion of eachxperiment to further confirm the depletion of CSPAs byTX treatment.2 TRPV1 immunoreactivity (Calbiochem,an Diego, Calif; 1:100) was performed by using an im-unohistochemistry procedure similar to that describedelow for Fos immunostaining, except that a fluores-ent-labeled secondary antibody was used. The numberf TRPV1-positive neurons in DRG and the area of TRPV1-ositive axons in spinal dorsal horn were calculated bysing an image analysis system. A total of 8 mice weresed for TRPV1 immunohistochemistry.

pinal Fos Expression and Fos–Tyrosineydroxylase Double Labeling in the LC

mmunohistochemistryIn the present study Fos immunohistochemistry waserformed on spinal cord tissue obtained 2 hoursost-BV injection because spinal cord Fos protein expres-ion typically reaches peak values at approximately 2ours after acute peripheral stimulation.9,40 Two hoursfter each dose of BV or saline injection (n � 5, respec-ively), animals were deeply anesthetized with 5% isoflu-ane and perfused transcardially with calcium-free Ty-ode’s solution followed by a fixative containing 4%araformaldehyde and 0.2% picric acid in 0.1 mol/Lhosphate buffer (pH 6.9). The spinal cord and brainere removed immediately after perfusion, post-fixed in

he same fixative for 4 hours, and then cryoprotected in0% sucrose in PBS for 48 hours (pH 7.4). Forty-microme-
er thick transverse frozen sections were cut through the t
pinal cord and brain by using a cryostat (Microm, Wall-orf, Germany).After elimination of endogenous peroxidase activityith 3% hydrogen peroxide in PBS and preblocking with% normal goat serum and 0.3% Triton X-100 in PBS,ections were incubated in polyclonal rabbit anti-Fos an-ibody (Calbiochem, EMD Biosciences; 1:10000) over-ight at 4°C. After several washes, the tissue sectionsere processed with the avidin-biotin method (EliteBC; Vector Laboratories, Burlingame, Calif). Finally, Fos

mmunoreactive neurons were visualized by using a 3,3=-iamino-benzidine (DAB; Sigma) reaction with 0.2%ickel chloride intensification (yielding black-labeledeuronal nuclei). For double labeling experiments to co-

ocalize Fos and tyrosine hydroxylase (TH, a marker ofatecholaminergic neuron as one of the catecholamineynthesis enzymes)13 in the LC region, the Fos-reactedections were thoroughly rinsed and subsequently incu-ated with rabbit anti-TH antibody (Biogenesis, Poole,ngland; 1:2000). TH immunoreactivity was visualized bysing a DAB reaction (yielding brown-labeled neuronalerikarya) as previously described.19

mage AnalysisAll data analysis procedures were performed blindlyith respect to the experimental condition of the ani-al. For quantitative analysis of Fos-positive neurons in

he lumbar spinal cord (L2-3) and LC region, sectionsere scanned, and then 5 spinal cord and 5 LC sectionsith the greatest number of Fos immunoreactive neu-

ons were selected from each animal. Spinal cord tissueections were first examined by using dark-field micros-opy (Zeiss Axioscope, Hallbergmoos, Germany) to de-ne the individual spinal cord laminae according to theray matter landmarks. The sections were then exam-

ned under a bright-field microscope at 100� to localizend quantify Fos-positive neurons. The L2-3 segments ofhe spinal cord were chosen for analysis in the presenttudy because these 2 segments receive primary afferentnput from the knee (Zusanli acupoint) area of the hindimb.34 Moreover, in a preliminary study we found thatV injection into the Zusanli acupoint selectively in-reased Fos expression in the L2-3 spinal cord segmentsather than the L4-6 segments, which receive input fromhe hind paw.To specifically identify the brainstem LC cell group, wesed the nomenclature and nuclear boundaries definedy Franklin and Paxinos in their stereotactic mouse braintlas. The region of the LC is located approximately1.50 mm to �1.95 mm behind the interaural line of therainstem. The selected sections were digitized with096 gray levels by using a cooled CCD (Micromax Kodak317; Princeton Instrument, Trenton, NJ) equipped withcomputer-assisted image analysis system (Metamorph;niversal Imaging Co, West Chester, Pa). To maintain aonstant threshold for each image and to compensateor subtle variability of immunostaining, we countednly neurons that were at least 30% darker than theverage gray level of each image after background sub-
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igure 1. Photomicrographs (A-D) of representative L2-L3 spi-al cord sections illustrating Fos expression in the dorsal hornfter administration of different doses of BV. Injection of salineA) or a low dose (0.001 mg/kg) of BV (B) induces very little Fosxpression in the dorsal horn. In contrast, administration of anntermediate dose (0.1 mg/kg) (C) or a high dose (10 mg/kg) (D)f BV produced a significant increase in spinal cord Fos expres-ion. Scale bar, 200 �m. (E, F) Graphs demonstrating the laminaristribution of Fos immunoreactive neurons in the ipsilateral (E)nd contralateral (F) dorsal horn (L2-3) induced by injection ofifferent doses of BV (n � 5 for all groups). *P � .05, **P � .01ignificantly different from the saline treatment group (SAL),
espectively. Total, entire spinal cord dorsal horn.
igure 2. Graphs illustrating the log dose-response curves forV’s effect on (A) the total counts of BV-induced Fos expression

n the entire spinal cord dorsal horn and (B) on formalin-inducedociceptive behavior during the second phase (10 to 30 minutesfter formalin injection) of the formalin test. The straight linesre derived from the equation Y � 13.23logX � 60.10 of thedministered dose with R � 0.945 in (A) and Y � �46.81logX �9.51 with R � 0.975 in (B). (C) A graph demonstrating the effectf BV injection (0.001, 0.1, and 10 mg/kg) into Zusanli point onpontaneous nociceptive behavior (0 to 60 minutes post-BV in-

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uced Fos staining was analyzed in the following 3 grayatter regions on the basis of cytoarchitectonic criteria:

1) the superficial dorsal horn (SDH, laminae I and II); (2)he nucleus proprius (NP, laminae III and IV); and (3) theeck region (NECK, laminae V and VI).Neurons double-labeled for Fos and TH were quanti-ed in the LC as previously described.19 Eight sectionshrough the LC were randomly selected from each ani-al and subsequently processed for Fos and TH double

abeling. The average number of immunoreactive neu-ons from each animal was calculated from at least 5epresentative sections. The percentage of Fos double-abeled catecholaminergic (TH) neurons was calculateds follows: Ratio of double labeling � Number of double-abeled (Fos and TH) neurons/Number of TH-labeled neu-ons � 100.

ormalin-Induced Pain Behavior TestTen minutes after BV injection, 1% formalin in a

igure 3. Graphs illustrating the antinociceptive effect pro-uced by injection of different doses (0.001-10 mg/kg) of BV onormalin-induced nociceptive behavior for the entire 30-minuteime course (A) and during the first (0-10 minutes) and secondhases (10-30 minutes) of the formalin test (B). *P � .05, **P �

01 significantly different from saline treatment group (SAL),espectively.

olume of 20 �L was injected subcutaneously into the m

lantar surface of the right hind paw with a 30-gaugeeedle. After formalin injection, the animals were im-ediately placed in an acrylic observation chamber (30

m in diameter and height), and nociceptive responsesn each animal were recorded by using a video cameraor a period of 30 minutes. The summation of time (ineconds) spent licking and biting the formalin-injectedind paw during each 5-minute block was measured asn indicator of the nociceptive response. Two experi-nced investigators who were blinded to the experi-ental conditions measured these formalin-induced

ehaviors. The duration of the responses during therst 10-minute period and the subsequent 10- to 30-inute period represents the first and second phases,

espectively, of the formalin test.To evaluate the nociceptive response induced by sub-

utaneous administration of different doses (0.001, 0.1,nd 10 mg/kg) of BV into the Zusanli acupoint in animalsithout formalin injection, the duration of spontaneousain behavior was measured for a period of 60 minutesfter injection by using the same method that was usedor the formalin test.

ntrathecal Injection of �2-AdrenoceptorntagonistTo evaluate the potential involvement of spinal �2-

drenoceptors on BVAN after RTX pretreatment, an �2-drenergic receptor antagonist, idazoxan (IDA, 10 �g/ice28; Sigma) was injected intrathecally 10 minutes

efore BV injection in both the SHAM and RTX-treatedroups. Six or 7 mice were randomly assigned to each BVr control group, respectively. Intrathecal injectionsere made by using a modification of the Hylden andilcox technique.10 Briefly, a 30-gauge needle con-

ected to a 50-�L Hamilton syringe with polyethyleneubing was inserted into the skin and then through the5-L6 intervertebral space directly into the subarachnoidpace. A flick of the mouse’s tail provided a reliable indi-ator that the needle had penetrated the dura, and 5 �Lf the drug was subsequently injected into the subarach-oid space.

tatistical AnalysisOne-way analysis of variance (ANOVA) was performed

o determine the overall effect of BV treatment on spinalos expression and on nociceptive behaviors as well as onhe resultant Fos-TH double labeling in the LC. An un-aired t test was used to determine the P value betweenhe vehicle (SHAM) and RTX-treatment groups, whereas

Newman-Keuls test was used to determine the 95%onfidence interval among the BV treatment groupshen ANOVA indicated a significant group difference. Avalue �.05 was considered statistically significant. All

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igure 4. Photomicrographs (A-D) and graphs (E, F) showing the effect of RTX treatment on capsaicin-sensitive neurons in aepresentative section through a DRG (B) and on capsaicin-sensitive axons in a representative section from the dorsal horn (D). ManyRPV1-ir neurons are evident in the DRG (A, E), whereas their central axonal processes are present in spinal dorsal horn (C, F) ofehicle-treated mice (SHAM, n � 8). Immunostaining is absent in the DRG (B, E) and dorsal horn (D, F) of mice that were treated withTX (n � 8). Scale bar, 200 �m. (G, H) Graphs demonstrating the effect of RTX treatment on the capsaicin-induced eye-wiping test (G,HAM: n � 24; RTX: n � 29) and on formalin-induced pain behavior (H, n � 9, respectively). RTX treatment totally suppressedapsaicin-induced eye-wiping behavior (**P � .01) and significantly reduced pain behavior during first phase (**P � .01), but not the
econd phase, of the formalin test.

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elationship Between the Dose of BVnd Its Nociceptive and AntinociceptiveffectsInjection of BV at doses ranging from 0.01 to 10 mg/kg

nto the right hind limb resulted in a significant dose-de-endent increase in Fos immunoreactive (Fos-ir) neurons inhe ipsilateral (right half, Fig 1A-E and Fig 2A), but not theontralateral (Fig 1F), dorsal horn of the lumbar spinal cord.njection of the 2 highest doses of BV (1 and 10 mg/kg)voked significant increases in Fos expression throughoutuch of the ipsilateral dorsal horn including the SDH, NP,

igure 5. Graphs illustrating the effect of vehicle (SHAM) orTX pretreatment on the antinociceptive effect produced by BV

njection (0.001-10 mg/kg) on formalin-induced nociceptive be-avior during the first phase (A, 0-10 minutes) and the secondhase (B, 10-30 minutes) of the formalin test (n �7 vehicle; n �RTX). *P � .05 and **P � .01 as compared with saline treat-ent, respectively.

nd NECK regions, whereas the intermediate doses of BV i

0.01 and 0.1 mg/kg) selectively increased Fos expressionnly in the SDH and NECK regions of the ipsilateral spinalorsal horn. Interestingly, all doses of BV (0.001, 0.1, and 10g/kg) injected into the Zusanli acupoint failed to evoke

ignificant nociceptive behaviors during the 60-minute ob-ervation period (Fig 2C). Thus BV injection did not produceny detectable nocifensive behaviors in comparison withhe vehicle treatment group.Similar to what was observed with BV-induced Fos ex-ression, injection of the lowest dose of BV (0.001 mg/kg)ad no suppressive effect on nociceptive behavior (paw

icking and biting time) in either the first or second phasef the formalin test (Fig 3A, B). Injection of the middleoses of BV produced a weak, nonsignificant antinoci-eptive effect, whereas injection of the high dose of BVroduced a significant antinociceptive effect on paw

icking/biting time during the first phase of the formalinest (Fig 3A, B). Although a dose-dependent effect wasot observed during the first phase, this could be anrtifact of the lower measures. In contrast, injection ofhe middle and high doses of BV (0.01, 0.1, 1, and 10g/kg) potently suppressed the second phase of forma-

in-induced pain as compared to the saline injection con-rol group, with the highest dose of BV producing a sig-ificantly more potent BVAN effect as compared to anyf the lower doses (Fig 2B and Fig 3A, B).

ffect of RTX Pretreatment on BV-nduced Spinal Fos Expression and BV-nduced AntinociceptionRTX treatment was found to dramatically suppress

ye-wiping behavior induced by dropping diluted cap-aicin (0.01%) onto the cornea in the majority of RTX-reated mice compared with non–RTX-treated miceFig 4G; P � .01). Furthermore, TRPV1-ir neurons thatre evident in the DRG and spinal cord dorsal horn ofehicle-treated mice (SHAM, Fig 4A, C) were not de-ected in the RTX-treated group (Fig 4B, D), furtherndicating that the RTX treatment was successful inepletion of CSPAs (Fig 4E, F; P � .01). It was notablehat RTX pretreatment itself significantly suppressedhe first phase of formalin-induced pain behavior butot the second phase of pain behavior (Fig 4H and FigA; P � .01). This result was consistent with those ofther previous studies.31, 41

Spinal Fos expression induced by the intermediateose of BV (0.1 mg/kg) was not affected by RTX pretreat-ent (Fig 6A, C and Fig 7). On the other hand, spinal Fos

xpression induced by the high dose of BV (10 mg/kg) inhe SDH and NECK regions was selectively attenuated byTX treatment (Fig 6B, D and Fig 7; P � .01 and P � .05,espectively), but the total number of Fos-ir neurons wasimilar to that of the intermediate-dose BV group (FigD). In addition, BV-induced Fos expression in the con-ralateral spinal cord dorsal horn was not affected byTX pretreatment (Fig 7E).On the other hand, RTX pretreatment did not reduce

he BVAN effect on the second phase of the formalin test
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reated groups (Fig 5B; P � .05 and P � .01). Similarly,TX pretreatment did not affect BVAN in the first phasef the formalin test in the high-dose BV treatment groupFig 5A; P � .01).

ffect of RTX Pretreatment on theeuronal Mechanism of the BV-Inducedntinociceptive EffectIn the vehicle groups (SHAM), BV treatments (0.1 and

0 mg/kg) significantly increased the number of Fos-xpressing neurons and the ratio of double-labeledos-TH immunoreactive neurons in the LC region (FigA-D; P � .01) as compared with the saline-treatedroup. This indicated that more TH-positive neurons

igure 6. Photomicrographs of representative spinal cord sectillustrating BV-induced Fos immunolabeling in the ipsilateral lumspinal cord section taken from an animal in the SHAM group txpression from an animal in the SHAM group treated with a higretreated with RTX followed by an injection of BV (0.1 mg/kgollowed by an injection of a higher dose of BV (10 mg/kg,). RTroduced by the 10-mg/kg dose of BV. Scale bar, 200 �m.

o-contained Fos immunoreactivity after BV treat- p

ent. This anatomic finding correlates well with thencreased antinociceptive effect produced by theseoses of BV (Fig 5). In the RTX pretreatment groups,he number of Fos immunoreactive neurons and theolocalization ratio between Fos and TH were nothanged in comparison to the SHAM groups (Fig 8C, D;

� .01), indicating that RTX had no effect on BV-nduced Fos expression in the LC.

Intrathecal idazoxan pretreatment (IDA, 10 �g/mice) inhe SHAM group (IDA-SAL) did not affect formalin-in-uced nociceptive behavior in comparison to intrathecalaline treatment in the SHAM group (SAL-SAL). On thether hand, IDA pretreatment blocked the developmentf BVAN produced by injection of either 0.1 or 10 mg/kgf BV (Fig 9). Importantly, the inhibitory effect producedy intrathecal IDA on BVAN was not affected by RTX

rom the vehicle (SHAM, A, B) and RTX (C, D) treatment groupsspinal cord dorsal horn. (A) Spinal Fos expression is illustrated inas treated with an intermediate dose (0.1 mg/kg) of BV. (B) Fosse of BV (10 mg/kg). (C) Spinal cord Fos expression in an animal) Spinal cord Fos expression in an animal pretreated with RTXtreatment caused a significant reduction in the Fos expression

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iscussioneripheral BV Stimulation–Induced Spinalos Expression Without Nociceptiveehavior Is Closely Related to BV’sntinociceptive EffectIt has been reported that intraplantar BV injection pro-uces a set of nocifensive behaviors including licking,iting, and flinching for a period of approximately 1our after injection.3,18 In contrast, we failed to detectny observable nocifensive behavior when different

igure 7. Graphs illustrating the effect of vehicle (SHAM) or RTXP, (C) the NECK, and in (D) the entire dorsal horn (Total-ipsi) fro

he effect of vehicle (SHAM) or RTX pretreatment on BV-inducedral spinal cord (n � 5). *P � .05 and **P � .01 as compared w

nduced spinal Fos expression was selectively attenuated by RTespectively).

oses of BV were injected into the Zusanli point in the h

resent as well as in previous studies.21 This differenceould be due to the fact that we are injecting BV directlynto an acupoint as opposed to a non-acupoint in theoot or to the fact that the subcutaneous tissue of theind paw has a greater innervation density than the areaear the stifle joint where the Zusanli acupoint is lo-ated. In addition, there are anatomic and likely func-ional differences between intraplantar glabrous skinnd the hairy skin where the Zusanli point is located inodents. Thus, these results indicate that BV stimulationf the Zusanli acupoint evokes very little nociceptive be-

treatment on BV-induced Fos expression in (A) the SDH, (B) thee ipsilateral spinal cord (n � 5, respectively). (E) Graph showingexpression in the entire dorsal horn (Total-contra) of contralat-aline treatment, respectively. The high dose of BV (10 mg/kg)-treatment in the SDH and NECK regions (P � .01 and P � .05,

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igure 8. Photomicrographs illustrating single- and double-labeled Fos and TH immunoreactive neurons in the LC region (A, B) andraphs (C, D) showing the effect of BV treatment on the number of Fos immunoreactive neurons in the LC region (C) and the ratiof Fos co-expression with TH (D) in either vehicle (SHAM) or RTX-pretreated mice (n � 5, respectively). The number of Fos/THouble-labeled neurons in animals treated with the high dose of BV (10 mg/kg) (B) was greater than that of saline-treated animalsA). **P � .01 compared with saline treatment group. White arrowhead, TH immunoreactive neuron; black arrowhead, Fos-labeled
euronal nuclei; white arrow, double-labeled neurons. Scale bar, 50 �m.

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an Zusanli acupoint might represent a more effectivecupuncture stimulation paradigm, it remains to be de-ermined whether BV injection into the human Zusanlicupoint is painful.On the other hand, spinal Fos expression, which served

s a marker of the neuronal activity induced by BV stim-lation of the Zusanli acupoint, was dose-dependently

ncreased particularly in the SDH region (Fig 1), withoses ranging from 0.001 mg/kg (which is typically notainful in human subjects) to 10 mg/kg (which is approx-

mately equivalent to the dose received in one honey-ee’s sting). Generally it is well-accepted that increases inociceptive stimulus intensity produce increases in dorsalorn Fos expression.8,11 However, the present studyemonstrates that the increase in spinal Fos expression

nduced by BV injection into the Zusanli acupoint did notorrelate with BV-induced nociceptive behavior. Therere 3 possible explanations for this discrepancy betweenpontaneous nociceptive behavior and spinal Fos expres-ion induced by BV injection into the Zusanli point. First,t is possible that the BV-induced Fos expression repre-ents a response to non-nociceptive stimulation at the BVnjection site, because not only noxious stimuli but alsonnocuous stimuli can produce spinal Fos expression.6,36

econd and perhaps more likely, BV injection into theusanli point does in fact produce nociception, but theevel of nociception, although great enough to evokepinal Fos expression, is not adequate to evoke detect-ble pain behaviors. Finally, it is possible that BV injectednto the Zusanli point does not produce observable no-iceptive behaviors (flinching, licking, or biting) as it doesn the hind paw, and although nociception was present,t was not measurable with the behavioral assays used inhe present study. We believe this latter explanation ishe most likely because we have also injected 1% forma-in together with BV into the Zusanli point and foundhat this combination failed to produce detectable noci-eptive behaviors (flinching, licking, or biting). Although

igure 9. Graph illustrating the effect of intrathecal (i.t.) salinend IDA (10 �g/mouse) on the BV (0.1 and 10 mg/kg)-inducedntinociceptive effect on the second phase (10-30 minutes) oformalin-induced pain behaviors in both vehicle (SHAM) andTX-pretreated groups (n � 6 and 7, respectively).

his might be due to differences in sensitivity between w

he hind paw and the subcutaneous tissue around thetifle joint, it is also possible that chemical activation ofhe Zusanli acupoint produces a profound antinocicep-ion without detectable nociception.We also showed that BVAN on pain behaviors associ-

ted with the second phase of the formalin test is alsoose-dependent and is produced by the same doses ofV (0.01, 0.1, 1, and 10 mg/kg) that produce spinal cordos expression (Fig 1B). These findings indicate that theagnitude of the BV-induced spinal neuronal activation

an be correlated with the magnitude of BVAN, suggest-ng that BVAN might result from a counter-irritation

echanism activated by peripheral stimulation. Al-hough counter-irritation is thought to be mediated byentral diffuse noxious inhibitory controls related to no-iceptive input,29 our findings importantly demon-trated that the BVAN can be produced without the in-uction of spontaneous nociceptive behavior.

ifferential Roles of Capsaicin-Sensitivefferents and Capsaicin-Insensitivefferents on BV-Induced Spinal Fosxpression and AntinociceptionAlthough RTX pretreatment did not alter the

mount of spinal Fos expression produced by eitherhe low or middle doses of BV, it selectively attenuatedhe increase in Fos expression evoked by the high dosef BV in both the SDH and NECK regions of the dorsalorn. (Figs 6 and 7) Importantly, RTX treatment did notffect the number of BV-induced Fos-ir neurons in theP region of the spinal cord dorsal horn. In addition,TX had no significant effect on BVAN (Fig 5). Thisould suggest that the high dose of BV either directlyr indirectly stimulates CSPAs, which in turn activateeurons in the SDH and NECK regions. This is consis-ent with previous anatomic studies demonstratinghat the central terminals of CSPAs primarily innervatehe SDH and NECK regions of the spinal cord, and thatheir activation by noxious heat or chemical stimuliesults in the induction of Fos protein in neurons in theDH and NECK, but not the NP, regions of the dorsalorn.12,26 This is also consistent with the present find-

ngs demonstrating that RTX pretreatment failed tolter the increase in Fos immunoreactive neurons in NPegion induced by the high dose of BV. This resultndicates that the high dose of BV probably activatesarge-diameter, low-threshold primary afferent neu-ons (mostly A fibers) in addition to small and me-ium afferents, and it also might serve to explain theotent analgesic effect of high-dose BV stimulationhat is thought to occur via the activation of spinalABAergic inhibitory interneurons.7

With respect to the type of primary afferent that istimulated, it has been reported that the threshold ofeuronal activation to electrical stimulation differs ac-ording to the type of primary afferent fiber (A, A,r C). For example, the minimum stimulus intensitiesnd durations required to activate C and A fibers
ere 110 �A, 0.4 millisecond and 34 �A, 0.1 millisec-

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nd, respectively, in an in vitro rat spinal cord prepa-ation.42 Furthermore, it has been shown that activa-ion of A fibers is most effective in producingrolonged inhibition of spinothalamic tract cells, al-hough significant additional effects were producedy stimulation of A�, A, and C fibers. These data,ogether with the findings of Uchida et al39 showinghat electroacupuncture stimulation causes an in-rease in spinal cord Fos expression via capsaicin-insen-itive primary afferent A fibers, suggest that the mostffective way to produce analgesia by peripheralerve stimulation would be by high-frequency stimu-

ation with an intensity strong enough to activate Abers.5,22 This concept is compatible with our resultshowing that activation of capsaicin-insensitive pri-ary afferents (CIPAs) by peripheral chemical stimula-

ion with diluted BV is most likely involved in BVAN.ecause most A afferents are insensitive to capsa-

cin,25 it is likely that BV is producing its antinocicep-ive effect by activation of A fibers at the site of in-ection.

ole of Capsaicin-Insensitive Afferents inhe Central Neuronal Mechanismsnderlying BV-Induced AntinociceptionWe have recently demonstrated that peripheral BV

njection increases Fos expression in rat brainstem cat-cholaminergic neurons including many neurons inhe LC.19 We have further shown that the activation ofpinal �2-adrenoceptors, but not opioid receptors, isritically involved in the BV-induced antinociceptivend antihyperalgesic effects observed in rodent mod-ls of visceral pain, inflammatory pain, and neuro-athic pain.16,17,30 As an extension of this work, the
resent study demonstrated that chemical stimulation f
eurobiol 77:299-352, 2005

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f the Zusanli acupoint by BV leads to activation oferipheral CIPAs fibers, which in turn evokes cat-cholaminergic neuronal activation in the LC region.ecause the coeruleospinal pathway plays an impor-ant role in the descending pain inhibitory system,38

ctivation of the LC by BV stimulation of CIPAs likelylays a key role in BV’s antinociceptive effects on for-alin-induced nociceptive behaviors. The fact that

TX pretreatment failed to alter the number of Fos-ireurons or the ratio of Fos-TH double-labeled neurons

n the LC induced by BV injection would support ourypothesis that BV injection activates the central nor-drenergic system via stimulation of peripheral CIPAs.his is further supported by the finding that intrathe-al pretreatment with the �2-adrenoceptor antagonistDA abolished BV’s antinociceptive effect on formalin-nduced pain behavior, whereas RTX pretreatmentailed to alter IDA’s inhibitory effect on BVAN. Collec-ively these data indicate that BV injection into theusanli acupoint stimulates peripheral CIPAs, which inurn activate catecholaminergic neurons in the LC. TheC then stimulates spinal cord �2-adrenoceptors viaescending noradrenergic pathways, and this leads toV’s antinociceptive effect on formalin-induced painehaviors.In conclusion, there are 2 important findings that stem

rom the data obtained in this study: (1) BV stimulationf the Zusanli acupoint produces a significant antinoci-eptive effect in the second phase of the formalin testhat involves spinal neuronal transmission without de-ectable nociceptive behavior, and (2) peripheral CIPAsre primarily involved in activating central catecholamin-rgic pathways associated with BV’s antinociceptive ef-
ect.
eferences

. Carlton SM, Zhou S, Kraemer B, Coggeshall RE: A role foreripheral somatostatin receptors in counter-irritation-in-uced analgesia. Neuroscience 120:499-508, 2003

. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA,evine JD, Julius D: The capsaicin receptor: A heat-activatedon channel in the pain pathway. Nature 389:816-824, 1997

. Chen J, Luo C, Li HL: The contribution of spinal neuronalhanges to development of prolonged, tonic nociceptiveesponses of the cat induced by subcutaneous bee venomnjection. Eur J Pain 2:359-376, 1998

. Chen J, Chen HS: Pivotal role of capsaicin-sensitive pri-ary afferents in development of both heat and mechanical

yperalgesia induced by intraplantar bee venom injection.ain 91:367-376, 2001

. Chung JM, Lee KH, Hori Y, Endo K, Willis WD: Factorsnfluencing peripheral nerve stimulation produced inhibi-ion of primate spinothalamic tract cells. Pain 19:277-293,984

. Coggeshall RE: Fos, nociception and the dorsal horn. Prog

. De Koninck Y, Henry JL: Prolonged GABAA-mediated in-ibition following single hair afferent input to single spinalorsal horn neurones in cats. J Physiol 476:89-100, 1994

. Doyle CA, Hunt SP: Substance P receptor (neurokinin-1)-xpressing neurons in lamina I of the spinal cord encode forhe intensity of noxious stimulation: a c-Fos study in rat.euroscience 89:17-28, 1999

. Fukura M, Kashima K, Maeda S, Shiba R: Changes in biteorce and muscle forces in the upper extremities afterounter irritation. Cranio 22:45-49, 2004

0. Hylden JL, Wilcox GL: Intrathecal morphine in mice: Aew technique. Eur J Pharmacol 67:313-316, 1980

1. Iwata K, Takahashi O, Tsuboi Y, Ochiai H, Hibiya J,akaki T, Yamaguchi Y, Sumino R: Fos protein induction inhe medullary dorsal horn and first segment of the spinalord by tooth-pulp stimulation in cats. Pain 75:27-36, 1998

2. Jinks SL, Simons CT, Dessirier JM, Carstens MI, AntogniniF, Carstens E: C-fos induction in rat superficial dorsal hornollowing cutaneous application of noxious chemical or me-hanical stimuli. Exp Brain Res 145:261-269, 2002

3. Jones SL: Descending noradrenergic influences on pain.
rog Brain Res 88:381-394, 1991

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4. Kim HW, Kwon YB, Ham TW, Roh DH, Yoon SY, Lee HJ,an HJ, Yang IS, Beitz AJ, Lee JH: Acupoint stimulation usingee venom attenuates formalin-induced pain behavior andpinal cord fos expression in rats. J Vet Med Sci 65:349-355,003

5. Kim HW, Kwon YB, Ham TW, Roh DH, Yoon SY, Kang SY,ang IS, Han HJ, Lee HJ, Beitz AJ, Lee JH: General pharma-ological profiles of bee venom and its water soluble frac-ions in rodent models. J Vet Sci 5:309-318, 2004

6. Kim HW, Kwon YB, Han HJ, Yang IS, Beitz AJ, Lee JH:ntinociceptive mechanisms associated with diluted beeenom acupuncture (apipuncture) in the rat formalin test:nvolvement of descending adrenergic and serotonergicathways. Pharmacol Res 51:183-188, 2005

7. Kwon YB, Kang MS, Han HJ, Beitz AJ, Lee JH: Visceralntinociception produced by bee venom stimulation of thehongwan acupuncture point in mice: Role of alpha(2) ad-enoceptors. Neurosci Lett 308:133-137, 2001

8. Kwon YB, Lee JD, Lee HJ, Han HJ, Mar WC, Kang SK,eitz AJ, Lee JH: Bee venom injection into an acupunctureoint reduces arthritis associated edema and nociceptiveesponses. Pain 90:271-280, 2001

9. Kwon YB, Han HJ, Beitz AJ, Lee JH: Bee venom acupointtimulation increases Fos expression in catecholaminergiceurons in the rat brain. Mol Cells 17:329-333, 2004

0. Lariviere WR, Melzack R: The bee venom test: Compari-ons with the formalin test with injection of different ven-ms. Pain 84:111-112, 2000

1. Lee JH, Kwon YB, Han HJ, Mar WC, Lee HJ, Yang IS, BeitzJ, Kang SK: Bee venom pretreatment has both an antino-iceptive and anti-inflammatory effect on carrageenan-in-uced inflammation. J Vet Med Sci 63:251-259, 2001

2. Lee KH, Chung JM, Willis WD Jr: Inhibition of primatepinothalamic tract cells by TENS. J Neurosurg 62:276-287,985

3. Luo C, Chen J, Li HL, Li JS: Spatial and temporal expres-ion of c-Fos protein in the spinal cord of anesthetized ratnduced by subcutaneous bee venom injection. Brain Res06:175-185, 1998

4. Mansikka H, Lahdesmaki J, Scheinin M, Pertovaara A:lpha2A adrenoceptors contribute to feedback inhibitionf capsaicin-induced hyperalgesia. Anesthesiology 101:185-90, 2004

5. Nagy JI, van der Kooy D: Effects of neonatal capsaicinreatment on nociceptive thresholds in the rat. J Neurosci:1145-1150, 1983

6. Nakatsuka T, Furue H, Yoshimura M, Gu JG: Activationf central terminal vanilloid receptor-1 receptors and alphaeta-methylene-ATP-sensitive P2X receptors reveals a con-erged synaptic activity onto the deep dorsal horn neuronsf the spinal cord. J Neurosci 22:1228-1237, 2002

7. Pan HL, Khan GM, Alloway KD, Chen SR: Resiniferatoxinnduces paradoxical changes in thermal and mechanical sen-itivities in rats: Mechanism of action. J Neurosci 23:2911-919, 2003

8. Pericic D: Swim stress inhibits 5-HT2A receptor-medi-ted head twitch behaviour in mice. Psychopharmacology
67:373-379, 2003 1
9. Roby-Brami A, Bussel B, Willer JC, Le Bars D: An electro-hysiological investigation into the pain-relieving effects ofeterotopic nociceptive stimuli: Probable involvement of aupraspinal loop. Brain 110:1497-1508, 1987

0. Roh DH, Kwon YB, Kim HW, Ham TW, Yoon SY, Kang SY,an HJ, Lee HJ, Beitz AJ, Lee JH: Acupoint stimulation withiluted bee venom (apipuncture) alleviates thermal hyper-lgesia in a rodent neuropathic pain model: involvement ofpinal alpha 2-adrenoceptors. J Pain 5:297-303, 2004

1. Shibata M, Ohkubo T, Takahashi H, Inoki R: Modifiedormalin test: Characteristic biphasic pain response. Pain 38:47-352, 1989

2. Szallasi A, Joo F, Blumberg PM: Duration of desensitiza-ion and ultrastructural changes in dorsal root ganglia inats treated with resiniferatoxin, an ultrapotent capsaicinnalog. Brain Res 503:68-72, 1989

3. Szolcsanyi J, Pinter E, Helyes Z, Oroszi G, Nemeth J: Sys-emic anti-inflammatory effect induced by counter-irrita-ion through a local release of somatostatin from nocicep-ors. Br J Pharmacol 125:916-922, 1998

4. Takahashi Y, Chiba T, Kurokawa M, Aoki Y: Dermatomesnd the central organization of dermatomes and body sur-ace regions in the spinal cord dorsal horn in rats. J Compeurol 462:29-41, 2003

5. Tender GC, Walbridge S, Olah Z, Karai L, Iadarola M,ldfield EH, Lonser RR: Selective ablation of nociceptiveeurons for elimination of hyperalgesia and neurogenic in-ammation. J Neurosurg 102:522-525, 2005

6. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gil-ert H, Skinner K, Raumann BE, Basbaum AI, Julius D: Theloned capsaicin receptor integrates multiple pain-producingtimuli. Neuron 21:531-543, 1998

7. Torsney C, Meredith-Middleton J, Fitzgerald M: Neona-al capsaicin treatment prevents the normal postnatal with-rawal of A fibres from lamina II without affecting fos re-ponses to innocuous peripheral stimulation. Brain Res Devrain Res 121:55-65, 2000

8. Tsuruoka M, Maeda M, Nagasawa I, Inoue T: Spinalathways mediating coeruleospinal antinociception in theat. Neurosci Lett 362:236-239, 2004

9. Uchida Y, Nishigori A, Takeda D, Ohshiro M, Ueda Y,hshima M, Kashiba H: Electroacupuncture induces the ex-ression of Fos in rat dorsal horn via capsaicin-insensitivefferents. Brain Res 978:136-140, 2003

0. Williams S, Evan GI, Hunt SP: Changing patterns of c-fosnduction in spinal neurons following thermal cutaneoustimulation in the rat. Neuroscience 36:73-81, 1990

1. Wismer CT, Faltynek CR, Jarvis MF, McGaraughty S: Dis-inct neurochemical mechanisms are activated following ad-inistration of different P2X receptor agonists into theindpaw of a rat. Brain Res 965:187-193, 2003

2. Yajiri Y, Yoshimura M, Okamoto M, Takahashi H, Hi-ashi H: A novel slow excitatory postsynaptic current in sub-tantia gelatinosa neurons of the rat spinal cord in vitro.euroscience 76:673-688, 1997

3. Zimmermann M: Ethical guidelines for investigations ofxperimental pain in conscious animals. Pain 16:109-110,
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bee venom injection significantly reduces nociceptive behavior in the mouse formalin test via...

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