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Bee Venom Injection Significantly Reduces Nociceptive Behavior

in the Mouse Formalin Test via Capsaicin-Insensitive Afferents

Dae-Hyun Roh,* Hyun-Woo Kim,* Seo-Yeon Yoon,* Seuk-Yun Kang,*Young-Bae Kwon, Kwang-Hyun Cho, Ho-Jae Han, Yeon-Hee Ryu, Sun-Mi Choi,

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

Department of Medical Research, Korea Institute of Oriental Medicine, Daejeon, South Korea.Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyunghee University, Seoul, SouthKorea.#Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, StPaul, 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 by

BV injection into the Zusanli acupoint is involved in producing the more prolonged antinociceptive

effect observed in the mouse formalin test, and whether capsaicin-sensitive primary afferents are

involved in these effects. BV injection into the Zusanli point increased spinal Fos expression but not

spontaneous nociceptive behavior. BV pretreatment 10 minutes before intraplantar formalin injection

dose-dependently attenuated nociceptive behavior associated with the second phase of the formalintest. The destruction of capsaicin-sensitive primary afferents by resiniferatoxin (RTX) pretreatment

selectively decreased BV-induced spinal Fos expression but did not affect BV-induced antinociception.

Furthermore, BV injection increased Fos expression in tyrosine hydroxylase immunoreactive neurons

in the locus caeruleus, and this expression was unaltered by RTX pretreatment. Finally, BVs antino-

ciception was blocked by intrathecal injection of 10 g idazoxan, and this effect was not modified by

RTX pretreatment. These findings suggest that subcutaneous BV stimulation of the Zusanli point

activates 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, which

ultimately 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 involves

activation 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.

Received May 10, 2005; Revised February 3, 2006; Accepted February 4,2006.

Supported by a grant (M103KV010009 03K2201 00940) from the BrainResearch Centerof the21st Century Frontier Research Program fundedbythe Ministry of Science and Technology of the Republic of Korea and bySRC program of KOSEF (R11-2005-014) as well as the Brain Korea 21

project.

Address requests forreprintsto Jang-HernLee, DVM, PhD, Department ofVeterinary Physiology, College of Veterinary Medicine, Seoul NationalUniversity, Seoul 151-742, South Korea. E-mail: [email protected]

1526-5900/$32.00

2006 by the American Pain Society

doi:10.1016/j.jpain.2006.02.002

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

500

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

ducing substances including melittin, histamine, andphospholipase A2, and therefore it is not surprising thatseveral reports described a nociceptive effect after in-traplantar injection.3,18,20 Luo et al23 have also reported

that intraplantar BV injection significantly increases Fosexpression in the spinal cord dorsal horn of anesthetizedrats. In contrast, subcutaneous injection of diluted BVinto an acupoint, termed apipuncture, has been usedclinically in Oriental medicine to produce a potent anal-gesic effect. In support of this alternative medicine ap-proach recent experimental studies in our laboratorieshave demonstrated that subcutaneous injection of BV(0.01 to 1 mg/kg) into the Zusanli acupuncture point pro-duces prominent antinociceptive and antihyperalgesiceffects in animal models of acute and persistent pain,respectively.15,17,18,30 The above studies indicate that adichotomy exists with respect to the physiologic re-

sponse to subcutaneous injection of BV. On one hand,intraplantar injection of BV and its major constituent,melittin, produces robust nociceptive behaviors and hy-persensitivity in rodents, whereas BV injection into theZusanli acupoint, on the other hand, produces little no-ciceptive behaviors, but rather a significant antinocicep-tive effect in a variety of animal pain models. Despite theapparent conflicting data in the literature regarding theconsequences of BV injection, there have been no studiesthat have examined a possible relationship between BVsnociceptive and antinociceptive effects, particularly withrespect to BV injection into an acupoint. To begin toexamine this issue, we evaluated whether the intensity

of the BV-induced nociception (as measured by sponta-neous pain behavior) and BV-induced neuronal activa-tion (as measured by spinal Fos expression) produced byinjection into the Zusanli acupoint is correlated with theintensity of the BV-induced antinociception (BVAN) inthe mouse formalin test. Because both BVs nociceptiveand antinociceptive effects appear to involve activationof primary afferent fibers, we also explored whether pri-mary afferent axons expressing the vanilloid receptor 1(TRPV1) were involved in either of these effects.

TRPV1-expressing primary afferent neurons, termedcapsaicin-sensitive primary afferents (CSPAs), have beenrecognized as nociceptive polymodal C-fibers whose cell

bodies are located in dorsal root ganglia. Functionally,CSPAs are known to play a major role in nociceptivetransmission.2,37 Recent studies with a BV-induced painmodel have shown that CSPAs play a critical role in me-diating both the thermal and mechanical hyperalgesiainduced by BV injection.4 On the other hand, capsaicin-induced excitation of TRPV1 receptors has also beenshown to be involved in counter-irritation mechanisms(ie, pain in one part of the body can be used to controlpain in another part) that are involved in inhibiting thedevelopment of subsequent nociceptive behaviors andinflammatory reactions at distant body sites in therat.1,33 On the basis of these studies, we hypothesized

that BV activation of CSPAs not only elicits a nociceptive

signal, but that activation of CSPAs can simultaneouslyproduce BVAN via a counter-irritation mechanism thatinvolves activation of the descending pain inhibitory sys-tem (DPIS). To test this hypothesis we examined whetherthe depletion of CSPAs by using resiniferatoxin (RTX)pretreatment35 could modify BV-induced spinal Fos ex-pression and BVAN in the formalin test.

BVAN involves activation of primary afferent fibers asdiscussed above. We have previously reported that BVANis blocked by intrathecal pretreatment with the 2-adre-noceptor antagonists idazoxan or yohimbine in severaldifferent pain models.16,17,30 This implies that BVAN ismediated by the activation of spinal 2-adrenoceptors,which are known to be involved with the DPIS.24 In thisregard, we have recently shown that peripheral BV injec-tion effectively increases brainstem catecholaminergicneuronal activity including the activity of the locus caer-uleus (LC).19 Therefore, the final objective of this studywas to evaluate whether RTX pretreatment also affectsBV-induced catecholaminergic neuronal activity in the

LC and subsequent spinal 2-adrenoceptor activation.

Materials and Methods

AnimalsExperiments were performed on male ICR mice weigh-

ing 20 to 25 g. All experimental animals were obtainedfrom the Laboratory Animal Center of Seoul NationalUniversity. They were housed in colony cages with freeaccess to food and water and maintained in tempera-ture- and light-controlled rooms (23C 2C, 12/12-hourlight/dark cycle with lights on at 7:00 AM) for at least 1

week before the study. All of the methods used in thepresent study were approved by the Animal Care and UseCommittee at Seoul National University and conform toNational Institutes of Health guidelines (NIH publicationno. 86-23, revised 1985). In addition, the ethical guide-lines for investigating experimental pain in conscious an-imals recommended by the International Association forthe Study of Pain were followed.43

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

acupoint on spinal Fos expression and nociceptive behav-iorsas well as on BVANin the formalin testin naive mice,

BV from Apis mellifera (Sigma, St Louis, Mo) was dis-solved in physiologic saline (20L) at doses ranging from0.001 to 10 mg/kg. A therapeutic dose of 0.005 to 0.5mg/kg of BV is typically used to produce analgesia inhuman patients and is considered to be safe because thisdose range does not appear to affect the central ner-vous, cardiovascular, respiratory, and gastrointestinalsystems.14 Accordingly, the dose range of BV used in thepresent study encompassed these clinically used doses.Diluted BV was subcutaneously administered into theZusanli acupoint of the right hind limb located on thelateral side of the stifle joint adjacent to the anteriortubercle of the tibia as previously described.16 Animals in

the control group received an injection of vehicle into

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the Zusanli acupoint. Five mice per individual groupwere used for analysis of the effects of different doses ofBV on spinal cord Fos expression and on BV-induced no-ciceptive behavior. Eight or more mice were included ineach BV treatment or control group for behavioral anal-ysis in the formalin test.

To evaluate the potential role of CSPAs in BV-induced

spinal Fos expression and on BVAN in the formalin test,only the high (10 mg/kg), middle (0.1 mg/kg), and low(0.001 mg/kg) doses of BV were used in these experi-ments. The extremely potent capsaicin analog RTX (0.2mg/kg; Sigma) was dissolved in a mixture of 10% Tween80, 10% ethanol, and 80% normal saline.12,27 Either RTXor vehicle (10% Tween 80, 10% ethanol, and 80% nor-mal saline; SHAM) was injected subcutaneously in a vol-ume of 50 L into the scruff of the neck of the mouseanesthetized with 3% isoflurane in a mixture of N2O/O2gas 2 weeks before performing BV-induced Fos immuno-histochemistry and the formalin test. We waited 2 weeksafter RTX pretreatment to test the possible role of CSPAs,

which is based on the timeframe used in a previousstudy.32 To confirm that RTX treatment destroyed CSPAs,a diluted capsaicin solution (0.01%, dissolved in saline)was dropped into cornea, and then the number of eyewipes was counted for 1 minute on the day before BV-induced Fos immunohistochemistry and formalin injec-tion (SHAM, n 24; RTX, n 29). In addition to countingcapsaicin-induced eye wipes, TRPV1 immunohistochem-istry was performed on both the dorsal root ganglion(DRG) and the spinal cord at the completion of eachexperiment to further confirm the depletion of CSPAs byRTX treatment.2 TRPV1 immunoreactivity (Calbiochem,San Diego, Calif; 1:100) was performed by using an im-

munohistochemistry procedure similar to that describedbelow for Fos immunostaining, except that a fluores-cent-labeled secondary antibody was used. The numberof TRPV1-positive neurons in DRG and the area of TRPV1-positive axons in spinal dorsal horn were calculated byusing an image analysis system. A total of 8 mice wereused for TRPV1 immunohistochemistry.

Spinal Fos Expression and FosTyrosineHydroxylase Double Labeling in the LC

Immunohistochemistry

In the present study Fos immunohistochemistry was

performed on spinal cord tissue obtained 2 hourspost-BV injection because spinal cord Fos protein expres-sion typically reaches peak values at approximately 2hours after acute peripheral stimulation.9,40 Two hoursafter each dose of BV or saline injection (n 5, respec-tively), animals were deeply anesthetized with 5% isoflu-rane and perfused transcardially with calcium-free Ty-rodes solution followed by a fixative containing 4%paraformaldehyde and 0.2% picric acid in 0.1 mol/Lphosphate buffer (pH 6.9). The spinal cord and brainwere removed immediately after perfusion, post-fixed inthe same fixative for 4 hours, and then cryoprotected in30% sucrose in PBS for 48 hours (pH 7.4). Forty-microme-

ter thick transverse frozen sections were cut through the

spinal cord and brain by using a cryostat (Microm, Wall-dorf, Germany).

After elimination of endogenous peroxidase activitywith 3% hydrogen peroxide in PBS and preblocking with3% normal goat serum and 0.3% Triton X-100 in PBS,sections were incubated in polyclonal rabbit anti-Fos an-tibody (Calbiochem, EMD Biosciences; 1:10000) over-

night at 4C. After several washes, the tissue sectionswere processed with the avidin-biotin method (EliteABC; Vector Laboratories, Burlingame, Calif). Finally, Fosimmunoreactive neurons were visualized by using a 3,3=-diamino-benzidine (DAB; Sigma) reaction with 0.2%nickel chloride intensification (yielding black-labeledneuronal nuclei). For double labeling experiments to co-localize Fos and tyrosine hydroxylase (TH, a marker ofcatecholaminergic neuron as one of the catecholaminesynthesis enzymes)13 in the LC region, the Fos-reactedsections were thoroughly rinsed and subsequently incu-bated with rabbit anti-TH antibody (Biogenesis, Poole,England; 1:2000). TH immunoreactivity was visualized by

using a DAB reaction (yielding brown-labeled neuronalperikarya) as previously described.19

Image Analysis

All data analysis procedures were performed blindlywith respect to the experimental condition of the ani-mal. For quantitative analysis of Fos-positive neurons inthe lumbar spinal cord (L2-3) and LC region, sectionswere scanned, and then 5 spinal cord and 5 LC sectionswith the greatest number of Fos immunoreactive neu-rons were selected from each animal. Spinal cord tissuesections were first examined by using dark-field micros-copy (Zeiss Axioscope, Hallbergmoos, Germany) to de-

fine the individual spinal cord laminae according to thegray matter landmarks. The sections were then exam-ined under a bright-field microscope at 100 to localizeand quantify Fos-positive neurons. The L2-3 segments ofthe spinal cord were chosen for analysis in the presentstudy because these 2 segments receive primary afferentinput from the knee (Zusanli acupoint) area of the hindlimb.34 Moreover, in a preliminary study we found thatBV injection into the Zusanli acupoint selectively in-creased Fos expression in the L2-3 spinal cord segmentsrather than the L4-6 segments, which receive input fromthe hind paw.

To specifically identify the brainstem LC cell group, we

used the nomenclature and nuclear boundaries definedby Franklin and Paxinos in their stereotactic mouse brainatlas. The region of the LC is located approximately1.50 mm to1.95 mm behind the interaural line of thebrainstem. The selected sections were digitized with4096 gray levels by using a cooled CCD (Micromax Kodak1317; Princeton Instrument, Trenton, NJ) equipped witha computer-assisted image analysis system (Metamorph;Universal Imaging Co, West Chester, Pa). To maintain aconstant threshold for each image and to compensatefor subtle variability of immunostaining, we countedonly neurons that were at least 30% darker than theaverage gray level of each image after background sub-

traction and shading correction were performed. BV-in-

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Figure 1. Photomicrographs (A-D) of representative L2-L3 spi-nal cord sections illustrating Fos expression in the dorsal hornafter administration of different doses of BV. Injection of saline(A) or a low dose (0.001 mg/kg) of BV (B) induces very little Fosexpression in the dorsal horn. In contrast, administration of anintermediate dose (0.1 mg/kg) (C) or a high dose (10 mg/kg) (D)of BV produced a significant increase in spinal cord Fos expres-sion. Scale bar, 200m. (E, F) Graphs demonstrating the laminardistribution of Fos immunoreactive neurons in the ipsilateral (E)and contralateral (F) dorsal horn (L2-3) induced by injection of

different doses of BV (n 5 for all groups). *P .05, **P .01significantly different from the saline treatment group (SAL),respectively. Total, entire spinal cord dorsal horn.

Figure 2. Graphs illustrating the log dose-response curves forBVs effect on (A) the total counts of BV-induced Fos expressioninthe entirespinalcorddorsalhornand (B) on formalin-inducednociceptive behavior during the second phase (10 to 30 minutesafter formalin injection) of the formalin test. The straight linesare derived from the equation Y 13.23logX 60.10 of theadministered dose with R 0.945 in (A) and Y 46.81logX89.51 with R 0.975 in (B). (C) A graph demonstrating theeffectof BV injection (0.001, 0.1, and 10 mg/kg) into Zusanli point onspontaneous nociceptive behavior (0 to 60 minutes post-BV in-

jection) in animals that did not receive formalin injection.


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duced Fos staining was analyzed in the following 3 graymatter regions on the basis of cytoarchitectonic criteria:(1) the superficial dorsal horn (SDH, laminae I and II); (2)the nucleus proprius (NP, laminae III and IV); and (3) theneck region (NECK, laminae V and VI).

Neurons double-labeled for Fos and TH were quanti-

fied in the LC as previously described.19 Eight sectionsthrough the LC were randomly selected from each ani-mal and subsequently processed for Fos and TH doublelabeling. The average number of immunoreactive neu-rons from each animal was calculated from at least 5representative sections. The percentage of Fos double-labeled catecholaminergic (TH) neurons was calculatedas follows: Ratio of double labelingNumber of double-labeled (Fos and TH) neurons/Number of TH-labeled neu-rons 100.

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

volume of 20 L was injected subcutaneously into the

plantar surface of the right hind paw with a 30-gauge

needle. After formalin injection, the animals were im-

mediately placed in an acrylic observation chamber (30

cm in diameter and height), and nociceptive responses

in each animal were recorded by using a video camera

for a period of 30 minutes. The summation of time (in

seconds) spent licking and biting the formalin-injected

hind paw during each 5-minute block was measured asan indicator of the nociceptive response. Two experi-

enced investigators who were blinded to the experi-

mental conditions measured these formalin-induced

behaviors. The duration of the responses during the

first 10-minute period and the subsequent 10- to 30-

minute period represents the first and second phases,

respectively, of the formalin test.

To evaluate the nociceptive response induced by sub-

cutaneous administration of different doses (0.001, 0.1,

and 10 mg/kg) of BV into the Zusanli acupoint in animals

without formalin injection, the duration of spontaneous

pain behavior was measured for a period of 60 minutesafter injection by using the same method that was used

for the formalin test.

Intrathecal Injection of2

-AdrenoceptorAntagonist

To evaluate the potential involvement of spinal 2-

adrenoceptors on BVAN after RTX pretreatment, an 2-

adrenergic receptor antagonist, idazoxan (IDA, 10 g/

mice28; Sigma) was injected intrathecally 10 minutes

before BV injection in both the SHAM and RTX-treated

groups. Six or 7 mice were randomly assigned to each BVor control group, respectively. Intrathecal injections

were made by using a modification of the Hylden and

Wilcox technique.10 Briefly, a 30-gauge needle con-

nected to a 50-L Hamilton syringe with polyethylene

tubing was inserted into the skin and then through the

L5-L6 intervertebral space directly into the subarachnoid

space. A flick of the mouses tail provided a reliable indi-

cator that the needle had penetrated the dura, and 5 L

of the drug was subsequently injected into the subarach-

noid space.

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

to determine the overall effect of BV treatment on spinal

Fos expression and on nociceptive behaviors as well as on

the resultant Fos-TH double labeling in the LC. An un-

paired ttest was used to determine the Pvalue between

the vehicle (SHAM) and RTX-treatment groups, whereas

a Newman-Keuls test was used to determine the 95%

confidence interval among the BV treatment groups

when ANOVA indicated a significant group difference. A

P value .05 was considered statistically significant. All

values are expressed as the mean standard error of the

mean.

Figure 3. Graphs illustrating the antinociceptive effect pro-duced by injection of different doses (0.001-10 mg/kg) of BV onformalin-induced nociceptive behavior for the entire 30-minutetime course (A) and during the first (0-10 minutes) and secondphases (10-30 minutes) of the formalin test (B). *P .05, **P.01 significantly different from saline treatment group (SAL),respectively.


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Figure 4. Photomicrographs (A-D) and graphs (E, F) showing the effect of RTX treatment on capsaicin-sensitive neurons in arepresentative section through a DRG (B) and on capsaicin-sensitive axons in a representative section from the dorsal horn (D). ManyTRPV1-ir neurons are evident in the DRG (A, E), whereas their central axonal processes are present in spinal dorsal horn ( C, F) ofvehicle-treated mice (SHAM, n 8). Immunostaining is absent in the DRG (B, E) and dorsal horn (D, F) of mice that were treated withRTX (n 8). Scale bar, 200 m. (G, H) Graphs demonstrating the effect of RTX treatment on the capsaicin-induced eye-wiping test (G,SHAM: n 24; RTX: n 29) and on formalin-induced pain behavior (H, n 9, respectively). RTX treatment totally suppressed

capsaicin-induced eye-wiping behavior (**P .01) and significantly reduced pain behavior during first phase (**P .01), but not thesecond phase, of the formalin test.


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Results

Relationship Between the Dose of BVand Its Nociceptive and AntinociceptiveEffects

Injection of BV at doses ranging from 0.01 to 10 mg/kginto the right hind limb resulted in a significant dose-de-pendent increase in Fos immunoreactive (Fos-ir) neurons inthe ipsilateral (right half, Fig 1A-E and Fig 2A), but not thecontralateral (Fig1F),dorsal horn of thelumbar spinalcord.Injection of the 2 highest doses of BV (1 and 10 mg/kg)evoked significant increases in Fos expression throughoutmuch of the ipsilateral dorsal horn including the SDH, NP,

and NECK regions, whereas the intermediate doses of BV

(0.01 and 0.1 mg/kg) selectively increased Fos expressiononly in the SDH and NECK regions of the ipsilateral spinaldorsalhorn. Interestingly, alldoses of BV (0.001, 0.1, and10mg/kg) injected into the Zusanli acupoint failed to evokesignificant nociceptive behaviors during the 60-minute ob-servation period (Fig2C).Thus BV injectiondidnot produceany detectable nocifensive behaviors in comparison with

the vehicle treatment group.Similar to what was observed with BV-induced Fos ex-

pression, injection of the lowest dose of BV (0.001 mg/kg)had no suppressive effect on nociceptive behavior (pawlicking and biting time) in either the first or second phaseof the formalin test (Fig 3A, B). Injection of the middledoses of BV produced a weak, nonsignificant antinoci-ceptive effect, whereas injection of the high dose of BVproduced a significant antinociceptive effect on pawlicking/biting time during the first phase of the formalintest (Fig 3A, B). Although a dose-dependent effect wasnot observed during the first phase, this could be anartifact of the lower measures. In contrast, injection of

the middle and high doses of BV (0.01, 0.1, 1, and 10mg/kg) potently suppressed the second phase of forma-lin-induced pain as compared to the saline injection con-trol group, with the highest dose of BV producing a sig-nificantly more potent BVAN effect as compared to anyof the lower doses (Fig 2B and Fig 3A, B).

Effect of RTX Pretreatment on BV-Induced Spinal Fos Expression and BV-Induced Antinociception

RTX treatment was found to dramatically suppresseye-wiping behavior induced by dropping diluted cap-

saicin (0.01%) onto the cornea in the majority of RTX-treated mice compared with nonRTX-treated mice(Fig 4G; P .01). Furthermore, TRPV1-ir neurons thatare evident in the DRG and spinal cord dorsal horn ofvehicle-treated mice (SHAM, Fig 4A, C) were not de-tected in the RTX-treated group (Fig 4B, D), furtherindicating that the RTX treatment was successful indepletion of CSPAs (Fig 4E, F; P .01). It was notablethat RTX pretreatment itself significantly suppressedthe first phase of formalin-induced pain behavior butnot the second phase of pain behavior (Fig 4H and Fig5A; P .01). This result was consistent with those ofother previous studies.31, 41

Spinal Fos expression induced by the intermediatedose of BV (0.1 mg/kg) was not affected by RTX pretreat-ment (Fig 6A, C and Fig 7). On the other hand, spinal Fosexpression induced by the high dose of BV (10 mg/kg) inthe SDH and NECK regions was selectively attenuated byRTX treatment (Fig 6B, D and Fig 7; P .01 and P .05,respectively), but the total number of Fos-ir neurons wassimilar to that of the intermediate-dose BV group (Fig7D). In addition, BV-induced Fos expression in the con-tralateral spinal cord dorsal horn was not affected byRTX pretreatment (Fig 7E).

On the other hand, RTX pretreatment did not reducethe BVAN effect on the second phase of the formalin test

in either the middle- or high-dose BV (0.1 and 10 mg/kg)

Figure 5. Graphs illustrating the effect of vehicle (SHAM) orRTX pretreatment on the antinociceptive effect produced by BVinjection (0.001-10 mg/kg) on formalin-induced nociceptive be-havior during the first phase (A, 0-10 minutes) and the secondphase (B, 10-30 minutes) of the formalin test (n 7 vehicle; n 9 RTX). *P .05 and **P .01 as compared with saline treat-ment, respectively.


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treated groups (Fig 5B; P .05 and P .01). Similarly,RTX pretreatment did not affect BVAN in the first phaseof the formalin test in the high-dose BV treatment group

(Fig 5A; P .01).

Effect of RTX Pretreatment on theNeuronal Mechanism of the BV-Induced

Antinociceptive EffectIn the vehicle groups (SHAM), BV treatments (0.1 and

10 mg/kg) significantly increased the number of Fos-expressing neurons and the ratio of double-labeledFos-TH immunoreactive neurons in the LC region (Fig8A-D; P .01) as compared with the saline-treatedgroup. This indicated that more TH-positive neurons

co-contained Fos immunoreactivity after BV treat-

ment. This anatomic finding correlates well with the

increased antinociceptive effect produced by these

doses of BV (Fig 5). In the RTX pretreatment groups,

the number of Fos immunoreactive neurons and thecolocalization ratio between Fos and TH were not

changed in comparison to the SHAM groups (Fig 8C, D;

P .01), indicating that RTX had no effect on BV-

induced Fos expression in the LC.

Intrathecal idazoxan pretreatment (IDA, 10g/mice) in

the SHAM group (IDA-SAL) did not affect formalin-in-

duced nociceptive behavior in comparison to intrathecal

saline treatment in the SHAM group (SAL-SAL). On the

other hand, IDA pretreatment blocked the development

of BVAN produced by injection of either 0.1 or 10 mg/kg

of BV (Fig 9). Importantly, the inhibitory effect produced

by intrathecal IDA on BVAN was not affected by RTX

pretreatment (Fig 9).

Figure 6. Photomicrographs of representative spinal cord sections from the vehicle (SHAM, A, B) and RTX (C, D) treatment groupsillustrating BV-induced Fos immunolabeling in the ipsilateral lumbar spinal cord dorsal horn. (A) Spinal Fos expression is illustrated ina spinal cord section taken from an animal in the SHAM group that was treated with an intermediate dose (0.1 mg/kg) of BV. ( B) Fosexpression from an animal in the SHAM group treated with a high dose of BV (10 mg/kg). ( C) Spinal cord Fos expression in an animalpretreated with RTX followed by an injection of BV (0.1 mg/kg). (D) Spinal cord Fos expression in an animal pretreated with RTXfollowed by an injection of a higher dose of BV (10 mg/kg,). RTX pretreatment caused a significant reduction in the Fos expressionproduced by the 10-mg/kg dose of BV. Scale bar, 200 m.


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Discussion

Peripheral BV StimulationInduced SpinalFos Expression Without NociceptiveBehavior Is Closely Related to BVs

Antinociceptive EffectIt has been reported that intraplantar BV injection pro-

duces a set of nocifensive behaviors including licking,biting, and flinching for a period of approximately 1hour after injection.3,18 In contrast, we failed to detectany observable nocifensive behavior when different

doses of BV were injected into the Zusanli point in the

present as well as in previous studies.21 This difference

could be due to the fact that we are injecting BV directly

into an acupoint as opposed to a non-acupoint in the

foot or to the fact that the subcutaneous tissue of the

hind paw has a greater innervation density than the area

near the stifle joint where the Zusanli acupoint is lo-

cated. In addition, there are anatomic and likely func-

tional differences between intraplantar glabrous skin

and the hairy skin where the Zusanli point is located in

rodents. Thus, these results indicate that BV stimulation

of the Zusanli acupoint evokes very little nociceptive be-

havior in the rodent. Although BV injection into the hu-

Figure 7. Graphs illustrating the effect of vehicle (SHAM) or RTX pretreatment on BV-induced Fos expression in (A) the SDH, (B) theNP, (C) the NECK, and in (D) the entire dorsal horn (Total-ipsi) from the ipsilateral spinal cord (n 5, respectively). (E) Graph showingthe effect of vehicle (SHAM) or RTX pretreatment on BV-induced Fos expression in the entire dorsal horn (Total-contra) of contralat-eral spinal cord (n 5). *P .05 and **P .01 as compared with saline treatment, respectively. The high dose of BV (10 mg/kg)-induced spinal Fos expression was selectively attenuated by RTX pretreatment in the SDH and NECK regions ( P .01 and P .05,respectively).


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


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ond, respectively, in an in vitro rat spinal cord prepa-ration.42 Furthermore, it has been shown that activa-tion of A fibers is most effective in producingprolonged inhibition of spinothalamic tract cells, al-though significant additional effects were producedby stimulation of A, A, and C fibers. These data,together with the findings of Uchida et al 39 showing

that electroacupuncture stimulation causes an in-crease in spinal cord Fos expression via capsaicin-insen-sitive primary afferent A fibers, suggest that the mosteffective way to produce analgesia by peripheralnerve stimulation would be by high-frequency stimu-lation with an intensity strong enough to activate Afibers.5,22 This concept is compatible with our resultsshowing that activation of capsaicin-insensitive pri-mary afferents (CIPAs) by peripheral chemical stimula-tion with diluted BV is most likely involved in BVAN.Because most A afferents are insensitive to capsa-icin,25 it is likely that BV is producing its antinocicep-tive effect by activation of A fibers at the site of in-

jection.

Role of Capsaicin-Insensitive Afferents inthe Central Neuronal MechanismsUnderlying BV-Induced Antinociception

We have recently demonstrated that peripheral BVinjection increases Fos expression in rat brainstem cat-echolaminergic neurons including many neurons inthe LC.19 We have further shown that the activation ofspinal 2-adrenoceptors, but not opioid receptors, iscritically involved in the BV-induced antinociceptiveand antihyperalgesic effects observed in rodent mod-els of visceral pain, inflammatory pain, and neuro-pathic pain.16,17,30 As an extension of this work, thepresent study demonstrated that chemical stimulation

of the Zusanli acupoint by BV leads to activation of

peripheral CIPAs fibers, which in turn evokes cat-

echolaminergic neuronal activation in the LC region.

Because the coeruleospinal pathway plays an impor-

tant role in the descending pain inhibitory system, 38

activation of the LC by BV stimulation of CIPAs likely

plays a key role in BVs antinociceptive effects on for-

malin-induced nociceptive behaviors. The fact thatRTX pretreatment failed to alter the number of Fos-ir

neurons or the ratio of Fos-TH double-labeled neurons

in the LC induced by BV injection would support our

hypothesis that BV injection activates the central nor-

adrenergic system via stimulation of peripheral CIPAs.

This is further supported by the finding that intrathe-

cal pretreatment with the 2-adrenoceptor antagonist

IDA abolished BVs antinociceptive effect on formalin-

induced pain behavior, whereas RTX pretreatment

failed to alter IDAs inhibitory effect on BVAN. Collec-

tively these data indicate that BV injection into the

Zusanli acupoint stimulates peripheral CIPAs, which inturn activate catecholaminergic neurons in the LC. The

LC then stimulates spinal cord 2-adrenoceptors via

descending noradrenergic pathways, and this leads to

BVs antinociceptive effect on formalin-induced pain

behaviors.

In conclusion, there are 2 important findings that stem

from the data obtained in this study: (1) BV stimulation

of the Zusanli acupoint produces a significant antinoci-

ceptive effect in the second phase of the formalin test

that involves spinal neuronal transmission without de-

tectable nociceptive behavior, and (2) peripheral CIPAs

are primarily involved in activating central catecholamin-

ergic pathways associated with BVs antinociceptive ef-

fect.

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