Bee Venom Injection Significantly Reduces Nociceptive Behavior in the Mouse Formalin Test via Capsaicin-Insensitive Afferents

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

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,储 Sun-Mi Choi,储 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, and ‡ Department 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, South Korea. 储 Department of Medical Research, Korea Institute of Oriental Medicine, Daejeon, South Korea. ¶ Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyunghee University, Seoul, South Korea. # Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota.

Abstract: Peripheral bee venom (BV) administration produces 2 contrasting effects, nociception and antinociception. 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 formalin test. 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, BV’s antinociception 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 induction 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 without 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 acupuncture 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 Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea and by SRC program of KOSEF (R11-2005-014) as well as the Brain Korea 21 project.

500

Address requests for reprints to Jang-Hern Lee, DVM, PhD, Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, 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

ORIGINAL REPORT/Roh et al

B

ee venom (BV) injection can produce both an initial nociceptive effect and a prolonged antinociceptive effect. BV contains a number of potential pain-producing substances including melittin, histamine, and phospholipase A2, and therefore it is not surprising that several reports described a nociceptive effect after intraplantar injection.3,18,20 Luo et al23 have also reported that intraplantar BV injection significantly increases Fos expression in the spinal cord dorsal horn of anesthetized rats. In contrast, subcutaneous injection of diluted BV into an acupoint, termed apipuncture, has been used clinically in Oriental medicine to produce a potent analgesic effect. In support of this alternative medicine approach recent experimental studies in our laboratories have demonstrated that subcutaneous injection of BV (0.01 to 1 mg/kg) into the Zusanli acupuncture point produces prominent antinociceptive and antihyperalgesic effects in animal models of acute and persistent pain, respectively.15,17,18,30 The above studies indicate that a dichotomy exists with respect to the physiologic response to subcutaneous injection of BV. On one hand, intraplantar injection of BV and its major constituent, melittin, produces robust nociceptive behaviors and hypersensitivity in rodents, whereas BV injection into the Zusanli acupoint, on the other hand, produces little nociceptive behaviors, but rather a significant antinociceptive effect in a variety of animal pain models. Despite the apparent conflicting data in the literature regarding the consequences of BV injection, there have been no studies that have examined a possible relationship between BV’s nociceptive and antinociceptive effects, particularly with respect to BV injection into an acupoint. To begin to examine this issue, we evaluated whether the intensity of the BV-induced nociception (as measured by spontaneous pain behavior) and BV-induced neuronal activation (as measured by spinal Fos expression) produced by injection into the Zusanli acupoint is correlated with the intensity of the BV-induced antinociception (BVAN) in the mouse formalin test. Because both BV’s nociceptive and antinociceptive effects appear to involve activation of primary afferent fibers, we also explored whether primary afferent axons expressing the vanilloid receptor 1 (TRPV1) were involved in either of these effects. TRPV1-expressing primary afferent neurons, termed capsaicin-sensitive primary afferents (CSPAs), have been recognized as nociceptive polymodal C-fibers whose cell bodies are located in dorsal root ganglia. Functionally, CSPAs are known to play a major role in nociceptive transmission.2,37 Recent studies with a BV-induced pain model have shown that CSPAs play a critical role in mediating both the thermal and mechanical hyperalgesia induced by BV injection.4 On the other hand, capsaicininduced excitation of TRPV1 receptors has also been shown to be involved in counter-irritation mechanisms (ie, pain in one part of the body can be used to control pain in another part) that are involved in inhibiting the development of subsequent nociceptive behaviors and inflammatory reactions at distant body sites in the rat.1,33 On the basis of these studies, we hypothesized that BV activation of CSPAs not only elicits a nociceptive

501 signal, but that activation of CSPAs can simultaneously produce BVAN via a counter-irritation mechanism that involves activation of the descending pain inhibitory system (DPIS). To test this hypothesis we examined whether the depletion of CSPAs by using resiniferatoxin (RTX) pretreatment35 could modify BV-induced spinal Fos expression and BVAN in the formalin test. BVAN involves activation of primary afferent fibers as discussed above. We have previously reported that BVAN is blocked by intrathecal pretreatment with the ␣2-adrenoceptor antagonists idazoxan or yohimbine in several different pain models.16,17,30 This implies that BVAN is mediated by the activation of spinal ␣2-adrenoceptors, which are known to be involved with the DPIS.24 In this regard, we have recently shown that peripheral BV injection effectively increases brainstem catecholaminergic neuronal activity including the activity of the locus caeruleus (LC).19 Therefore, the final objective of this study was to evaluate whether RTX pretreatment also affects BV-induced catecholaminergic neuronal activity in the LC and subsequent spinal ␣2-adrenoceptor activation.

Materials and Methods Animals Experiments were performed on male ICR mice weighing 20 to 25 g. All experimental animals were obtained from the Laboratory Animal Center of Seoul National University. They were housed in colony cages with free access to food and water and maintained in temperature- and light-controlled rooms (23°C ⫾ 2°C, 12/12-hour light/dark cycle with lights on at 7:00 AM) for at least 1 week before the study. All of the methods used in the present study were approved by the Animal Care and Use Committee at Seoul National University and conform to National Institutes of Health guidelines (NIH publication no. 86-23, revised 1985). In addition, the ethical guidelines for investigating experimental pain in conscious animals recommended by the International Association for the Study of Pain were followed.43

BV Administration and RTX Pretreatment To evaluate the effect of BV injection into the Zusanli acupoint on spinal Fos expression and nociceptive behaviors as well as on BVAN in the formalin test in naive mice, BV from Apis mellifera (Sigma, St Louis, Mo) was dissolved in physiologic saline (20 ␮L) at doses ranging from 0.001 to 10 mg/kg. A therapeutic dose of 0.005 to 0.5 mg/kg of BV is typically used to produce analgesia in human patients and is considered to be safe because this dose range does not appear to affect the central nervous, cardiovascular, respiratory, and gastrointestinal systems.14 Accordingly, the dose range of BV used in the present study encompassed these clinically used doses. Diluted BV was subcutaneously administered into the Zusanli acupoint of the right hind limb located on the lateral side of the stifle joint adjacent to the anterior tubercle of the tibia as previously described.16 Animals in the control group received an injection of vehicle into

502 the Zusanli acupoint. Five mice per individual group were used for analysis of the effects of different doses of BV on spinal cord Fos expression and on BV-induced nociceptive behavior. Eight or more mice were included in each BV treatment or control group for behavioral analysis 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 experiments. The extremely potent capsaicin analog RTX (0.2 mg/kg; Sigma) was dissolved in a mixture of 10% Tween 80, 10% ethanol, and 80% normal saline.12,27 Either RTX or vehicle (10% Tween 80, 10% ethanol, and 80% normal saline; SHAM) was injected subcutaneously in a volume of 50 ␮L into the scruff of the neck of the mouse anesthetized with 3% isoflurane in a mixture of N2O/O2 gas 2 weeks before performing BV-induced Fos immunohistochemistry and the formalin test. We waited 2 weeks after RTX pretreatment to test the possible role of CSPAs, which is based on the timeframe used in a previous study.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 eye wipes was counted for 1 minute on the day before BVinduced Fos immunohistochemistry and formalin injection (SHAM, n ⫽ 24; RTX, n ⫽ 29). In addition to counting capsaicin-induced eye wipes, TRPV1 immunohistochemistry was performed on both the dorsal root ganglion (DRG) and the spinal cord at the completion of each experiment to further confirm the depletion of CSPAs by RTX treatment.2 TRPV1 immunoreactivity (Calbiochem, San Diego, Calif; 1:100) was performed by using an immunohistochemistry procedure similar to that described below for Fos immunostaining, except that a fluorescent-labeled secondary antibody was used. The number of TRPV1-positive neurons in DRG and the area of TRPV1positive axons in spinal dorsal horn were calculated by using an image analysis system. A total of 8 mice were used for TRPV1 immunohistochemistry.

Spinal Fos Expression and Fos–Tyrosine Hydroxylase Double Labeling in the LC Immunohistochemistry In the present study Fos immunohistochemistry was performed on spinal cord tissue obtained 2 hours post-BV injection because spinal cord Fos protein expression typically reaches peak values at approximately 2 hours after acute peripheral stimulation.9,40 Two hours after each dose of BV or saline injection (n ⫽ 5, respectively), animals were deeply anesthetized with 5% isoflurane and perfused transcardially with calcium-free Tyrode’s solution followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 mol/L phosphate buffer (pH 6.9). The spinal cord and brain were removed immediately after perfusion, post-fixed in the same fixative for 4 hours, and then cryoprotected in 30% sucrose in PBS for 48 hours (pH 7.4). Forty-micrometer thick transverse frozen sections were cut through the

Mechanism of Bee Venom–Induced Antinociception spinal cord and brain by using a cryostat (Microm, Walldorf, Germany). After elimination of endogenous peroxidase activity with 3% hydrogen peroxide in PBS and preblocking with 3% normal goat serum and 0.3% Triton X-100 in PBS, sections were incubated in polyclonal rabbit anti-Fos antibody (Calbiochem, EMD Biosciences; 1:10000) overnight at 4°C. After several washes, the tissue sections were processed with the avidin-biotin method (Elite ABC; Vector Laboratories, Burlingame, Calif). Finally, Fos immunoreactive neurons were visualized by using a 3,3=diamino-benzidine (DAB; Sigma) reaction with 0.2% nickel chloride intensification (yielding black-labeled neuronal nuclei). For double labeling experiments to colocalize Fos and tyrosine hydroxylase (TH, a marker of catecholaminergic neuron as one of the catecholamine synthesis enzymes)13 in the LC region, the Fos-reacted sections were thoroughly rinsed and subsequently incubated with rabbit anti-TH antibody (Biogenesis, Poole, England; 1:2000). TH immunoreactivity was visualized by using a DAB reaction (yielding brown-labeled neuronal perikarya) as previously described.19

Image Analysis All data analysis procedures were performed blindly with respect to the experimental condition of the animal. For quantitative analysis of Fos-positive neurons in the lumbar spinal cord (L2-3) and LC region, sections were scanned, and then 5 spinal cord and 5 LC sections with the greatest number of Fos immunoreactive neurons were selected from each animal. Spinal cord tissue sections were first examined by using dark-field microscopy (Zeiss Axioscope, Hallbergmoos, Germany) to define the individual spinal cord laminae according to the gray matter landmarks. The sections were then examined under a bright-field microscope at 100⫻ to localize and quantify Fos-positive neurons. The L2-3 segments of the spinal cord were chosen for analysis in the present study because these 2 segments receive primary afferent input from the knee (Zusanli acupoint) area of the hind limb.34 Moreover, in a preliminary study we found that BV injection into the Zusanli acupoint selectively increased Fos expression in the L2-3 spinal cord segments rather than the L4-6 segments, which receive input from the hind paw. To specifically identify the brainstem LC cell group, we used the nomenclature and nuclear boundaries defined by Franklin and Paxinos in their stereotactic mouse brain atlas. The region of the LC is located approximately ⫺1.50 mm to ⫺1.95 mm behind the interaural line of the brainstem. The selected sections were digitized with 4096 gray levels by using a cooled CCD (Micromax Kodak 1317; Princeton Instrument, Trenton, NJ) equipped with a computer-assisted image analysis system (Metamorph; Universal Imaging Co, West Chester, Pa). To maintain a constant threshold for each image and to compensate for subtle variability of immunostaining, we counted only neurons that were at least 30% darker than the average gray level of each image after background subtraction and shading correction were performed. BV-in-

ORIGINAL REPORT/Roh et al

503

Figure 2. Graphs illustrating the log dose-response curves for Figure 1. Photomicrographs (A-D) of representative L2-L3 spinal cord sections illustrating Fos expression in the dorsal horn after administration of different doses of BV. Injection of saline (A) or a low dose (0.001 mg/kg) of BV (B) induces very little Fos expression in the dorsal horn. In contrast, administration of an intermediate dose (0.1 mg/kg) (C) or a high dose (10 mg/kg) (D) of BV produced a significant increase in spinal cord Fos expression. Scale bar, 200 ␮m. (E, F) Graphs demonstrating the laminar distribution 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 ⬍ .01 significantly different from the saline treatment group (SAL), respectively. Total, entire spinal cord dorsal horn.

BV’s effect on (A) the total counts of BV-induced Fos expression in the entire spinal cord dorsal horn and (B) on formalin-induced nociceptive behavior during the second phase (10 to 30 minutes after formalin injection) of the formalin test. The straight lines are derived from the equation Y ⫽ 13.23logX ⫹ 60.10 of the administered dose with R ⫽ 0.945 in (A) and Y ⫽ ⫺46.81logX ⫹ 89.51 with R ⫽ 0.975 in (B). (C) A graph demonstrating the effect of BV injection (0.001, 0.1, and 10 mg/kg) into Zusanli point on spontaneous nociceptive behavior (0 to 60 minutes post-BV injection) in animals that did not receive formalin injection.

504

Mechanism of Bee Venom–Induced Antinociception plantar surface of the right hind paw with a 30-gauge needle. After formalin injection, the animals were immediately 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 as an indicator of the nociceptive response. Two experienced investigators who were blinded to the experimental conditions measured these formalin-induced behaviors. The duration of the responses during the first 10-minute period and the subsequent 10- to 30minute period represents the first and second phases, respectively, of the formalin test. To evaluate the nociceptive response induced by subcutaneous 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 minutes after injection by using the same method that was used for the formalin test.

Intrathecal Injection of ␣2-Adrenoceptor Antagonist

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

duced Fos staining was analyzed in the following 3 gray matter 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) the neck region (NECK, laminae V and VI). Neurons double-labeled for Fos and TH were quantified in the LC as previously described.19 Eight sections through the LC were randomly selected from each animal and subsequently processed for Fos and TH double labeling. The average number of immunoreactive neurons from each animal was calculated from at least 5 representative sections. The percentage of Fos doublelabeled catecholaminergic (TH) neurons was calculated as follows: Ratio of double labeling ⫽ Number of doublelabeled (Fos and TH) neurons/Number of TH-labeled neurons ⫻ 100.

Formalin-Induced Pain Behavior Test Ten minutes after BV injection, 1% formalin in a volume of 20 ␮L was injected subcutaneously into the

To evaluate the potential involvement of spinal ␣2adrenoceptors on BVAN after RTX pretreatment, an ␣2adrenergic 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 BV or control group, respectively. Intrathecal injections were made by using a modification of the Hylden and Wilcox technique.10 Briefly, a 30-gauge needle connected 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 mouse’s tail provided a reliable indicator that the needle had penetrated the dura, and 5 ␮L of the drug was subsequently injected into the subarachnoid space.

Statistical Analysis One-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 unpaired t test was used to determine the P value 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.

ORIGINAL REPORT/Roh et al

505

Figure 4. Photomicrographs (A-D) and graphs (E, F) showing the effect of RTX treatment on capsaicin-sensitive neurons in a representative section through a DRG (B) and on capsaicin-sensitive axons in a representative section from the dorsal horn (D). Many TRPV1-ir neurons are evident in the DRG (A, E), whereas their central axonal processes are present in spinal dorsal horn (C, F) of vehicle-treated mice (SHAM, n ⫽ 8). Immunostaining is absent in the DRG (B, E) and dorsal horn (D, F) of mice that were treated with RTX (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 the second phase, of the formalin test.

506

Mechanism of Bee Venom–Induced Antinociception (0.01 and 0.1 mg/kg) selectively increased Fos expression only in the SDH and NECK regions of the ipsilateral spinal dorsal horn. Interestingly, all doses of BV (0.001, 0.1, and 10 mg/kg) injected into the Zusanli acupoint failed to evoke significant nociceptive behaviors during the 60-minute observation period (Fig 2C). Thus BV injection did not produce any detectable nocifensive behaviors in comparison with the vehicle treatment group. Similar to what was observed with BV-induced Fos expression, injection of the lowest dose of BV (0.001 mg/kg) had no suppressive effect on nociceptive behavior (paw licking and biting time) in either the first or second phase of the formalin test (Fig 3A, B). Injection of the middle doses of BV produced a weak, nonsignificant antinociceptive effect, whereas injection of the high dose of BV produced a significant antinociceptive effect on paw licking/biting time during the first phase of the formalin test (Fig 3A, B). Although a dose-dependent effect was not observed during the first phase, this could be an artifact of the lower measures. In contrast, injection of the middle and high doses of BV (0.01, 0.1, 1, and 10 mg/kg) potently suppressed the second phase of formalin-induced pain as compared to the saline injection control group, with the highest dose of BV producing a significantly more potent BVAN effect as compared to any of the lower doses (Fig 2B and Fig 3A, B).

Effect of RTX Pretreatment on BVInduced Spinal Fos Expression and BVInduced Antinociception

Figure 5. Graphs illustrating the effect of vehicle (SHAM) or RTX pretreatment on the antinociceptive effect produced by BV injection (0.001-10 mg/kg) on formalin-induced nociceptive behavior during the first phase (A, 0-10 minutes) and the second phase (B, 10-30 minutes) of the formalin test (n ⫽7 vehicle; n ⫽ 9 RTX). *P ⬍ .05 and **P ⬍ .01 as compared with saline treatment, respectively.

Results Relationship Between the Dose of BV and Its Nociceptive and Antinociceptive Effects Injection of BV at doses ranging from 0.01 to 10 mg/kg into the right hind limb resulted in a significant dose-dependent increase in Fos immunoreactive (Fos-ir) neurons in the ipsilateral (right half, Fig 1A-E and Fig 2A), but not the contralateral (Fig 1F), dorsal horn of the lumbar spinal cord. Injection of the 2 highest doses of BV (1 and 10 mg/kg) evoked significant increases in Fos expression throughout much of the ipsilateral dorsal horn including the SDH, NP, and NECK regions, whereas the intermediate doses of BV

RTX treatment was found to dramatically suppress eye-wiping behavior induced by dropping diluted capsaicin (0.01%) onto the cornea in the majority of RTXtreated mice compared with non–RTX-treated mice (Fig 4G; P ⬍ .01). Furthermore, TRPV1-ir neurons that are evident in the DRG and spinal cord dorsal horn of vehicle-treated mice (SHAM, Fig 4A, C) were not detected in the RTX-treated group (Fig 4B, D), further indicating that the RTX treatment was successful in depletion of CSPAs (Fig 4E, F; P ⬍ .01). It was notable that RTX pretreatment itself significantly suppressed the first phase of formalin-induced pain behavior but not the second phase of pain behavior (Fig 4H and Fig 5A; P ⬍ .01). This result was consistent with those of other previous studies.31, 41 Spinal Fos expression induced by the intermediate dose of BV (0.1 mg/kg) was not affected by RTX pretreatment (Fig 6A, C and Fig 7). On the other hand, spinal Fos expression induced by the high dose of BV (10 mg/kg) in the SDH and NECK regions was selectively attenuated by RTX treatment (Fig 6B, D and Fig 7; P ⬍ .01 and P ⬍ .05, respectively), but the total number of Fos-ir neurons was similar to that of the intermediate-dose BV group (Fig 7D). In addition, BV-induced Fos expression in the contralateral spinal cord dorsal horn was not affected by RTX pretreatment (Fig 7E). On the other hand, RTX pretreatment did not reduce the BVAN effect on the second phase of the formalin test in either the middle- or high-dose BV (0.1 and 10 mg/kg)–

ORIGINAL REPORT/Roh et al

507

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

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

Effect of RTX Pretreatment on the Neuronal Mechanism of the BV-Induced Antinociceptive Effect In the vehicle groups (SHAM), BV treatments (0.1 and 10 mg/kg) significantly increased the number of Fosexpressing neurons and the ratio of double-labeled Fos-TH immunoreactive neurons in the LC region (Fig 8A-D; P ⬍ .01) as compared with the saline-treated group. 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 the colocalization 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 BVinduced Fos expression in the LC. Intrathecal idazoxan pretreatment (IDA, 10 ␮g/mice) in the SHAM group (IDA-SAL) did not affect formalin-induced 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).

508

Mechanism of Bee Venom–Induced Antinociception

Figure 7. Graphs illustrating the effect of vehicle (SHAM) or RTX pretreatment on BV-induced Fos expression in (A) the SDH, (B) the

NP, (C) the NECK, and in (D) the entire dorsal horn (Total-ipsi) from the ipsilateral spinal cord (n ⫽ 5, respectively). (E) Graph showing the effect of vehicle (SHAM) or RTX pretreatment on BV-induced Fos expression in the entire dorsal horn (Total-contra) of contralateral 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).

Discussion Peripheral BV Stimulation–Induced Spinal Fos Expression Without Nociceptive Behavior Is Closely Related to BV’s Antinociceptive Effect It has been reported that intraplantar BV injection produces a set of nocifensive behaviors including licking, biting, and flinching for a period of approximately 1 hour after injection.3,18 In contrast, we failed to detect any 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 located. In addition, there are anatomic and likely functional 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 behavior in the rodent. Although BV injection into the hu-

ORIGINAL REPORT/Roh et al

509

Figure 8. Photomicrographs illustrating single- and double-labeled Fos and TH immunoreactive neurons in the LC region (A, B) and graphs (C, D) showing the effect of BV treatment on the number of Fos immunoreactive neurons in the LC region (C) and the ratio of Fos co-expression with TH (D) in either vehicle (SHAM) or RTX-pretreated mice (n ⫽ 5, respectively). The number of Fos/TH double-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-labeled neuronal nuclei; white arrow, double-labeled neurons. Scale bar, 50 ␮m.

510

Figure 9. Graph illustrating the effect of intrathecal (i.t.) saline

and IDA (10 ␮g/mouse) on the BV (0.1 and 10 mg/kg)-induced antinociceptive effect on the second phase (10-30 minutes) of formalin-induced pain behaviors in both vehicle (SHAM) and RTX-pretreated groups (n ⫽ 6 and 7, respectively).

man Zusanli acupoint might represent a more effective acupuncture stimulation paradigm, it remains to be determined whether BV injection into the human Zusanli acupoint is painful. On the other hand, spinal Fos expression, which served as a marker of the neuronal activity induced by BV stimulation of the Zusanli acupoint, was dose-dependently increased particularly in the SDH region (Fig 1), with doses ranging from 0.001 mg/kg (which is typically not painful in human subjects) to 10 mg/kg (which is approximately equivalent to the dose received in one honeybee’s sting). Generally it is well-accepted that increases in nociceptive stimulus intensity produce increases in dorsal horn Fos expression.8,11 However, the present study demonstrates that the increase in spinal Fos expression induced by BV injection into the Zusanli acupoint did not correlate with BV-induced nociceptive behavior. There are 3 possible explanations for this discrepancy between spontaneous nociceptive behavior and spinal Fos expression induced by BV injection into the Zusanli point. First, it is possible that the BV-induced Fos expression represents a response to non-nociceptive stimulation at the BV injection site, because not only noxious stimuli but also innocuous stimuli can produce spinal Fos expression.6,36 Second and perhaps more likely, BV injection into the Zusanli point does in fact produce nociception, but the level of nociception, although great enough to evoke spinal Fos expression, is not adequate to evoke detectable pain behaviors. Finally, it is possible that BV injected into the Zusanli point does not produce observable nociceptive behaviors (flinching, licking, or biting) as it does in the hind paw, and although nociception was present, it was not measurable with the behavioral assays used in the present study. We believe this latter explanation is the most likely because we have also injected 1% formalin together with BV into the Zusanli point and found that this combination failed to produce detectable nociceptive behaviors (flinching, licking, or biting). Although this might be due to differences in sensitivity between

Mechanism of Bee Venom–Induced Antinociception the hind paw and the subcutaneous tissue around the stifle joint, it is also possible that chemical activation of the Zusanli acupoint produces a profound antinociception without detectable nociception. We also showed that BVAN on pain behaviors associated with the second phase of the formalin test is also dose-dependent and is produced by the same doses of BV (0.01, 0.1, 1, and 10 mg/kg) that produce spinal cord Fos expression (Fig 1B). These findings indicate that the magnitude of the BV-induced spinal neuronal activation can be correlated with the magnitude of BVAN, suggesting that BVAN might result from a counter-irritation mechanism activated by peripheral stimulation. Although counter-irritation is thought to be mediated by central diffuse noxious inhibitory controls related to nociceptive input,29 our findings importantly demonstrated that the BVAN can be produced without the induction of spontaneous nociceptive behavior.

Differential Roles of Capsaicin-Sensitive Afferents and Capsaicin-Insensitive Afferents on BV-Induced Spinal Fos Expression and Antinociception Although RTX pretreatment did not alter the amount of spinal Fos expression produced by either the low or middle doses of BV, it selectively attenuated the increase in Fos expression evoked by the high dose of BV in both the SDH and NECK regions of the dorsal horn. (Figs 6 and 7) Importantly, RTX treatment did not affect the number of BV-induced Fos-ir neurons in the NP region of the spinal cord dorsal horn. In addition, RTX had no significant effect on BVAN (Fig 5). This would suggest that the high dose of BV either directly or indirectly stimulates CSPAs, which in turn activate neurons in the SDH and NECK regions. This is consistent with previous anatomic studies demonstrating that the central terminals of CSPAs primarily innervate the SDH and NECK regions of the spinal cord, and that their activation by noxious heat or chemical stimuli results in the induction of Fos protein in neurons in the SDH and NECK, but not the NP, regions of the dorsal horn.12,26 This is also consistent with the present findings demonstrating that RTX pretreatment failed to alter the increase in Fos immunoreactive neurons in NP region induced by the high dose of BV. This result indicates that the high dose of BV probably activates large-diameter, low-threshold primary afferent neurons (mostly A␤ fibers) in addition to small and medium afferents, and it also might serve to explain the potent analgesic effect of high-dose BV stimulation that is thought to occur via the activation of spinal GABAergic inhibitory interneurons.7 With respect to the type of primary afferent that is stimulated, it has been reported that the threshold of neuronal activation to electrical stimulation differs according to the type of primary afferent fiber (A␤, A␦, or C). For example, the minimum stimulus intensities and durations required to activate C and A␦ fibers were 110 ␮A, 0.4 millisecond and 34 ␮A, 0.1 millisec-

ORIGINAL REPORT/Roh et al ond, respectively, in an in vitro rat spinal cord preparation.42 Furthermore, it has been shown that activation of A␦ fibers is most effective in producing prolonged inhibition of spinothalamic tract cells, although significant additional effects were produced by stimulation of A␣, A␤, and C fibers. These data, together with the findings of Uchida et al39 showing that electroacupuncture stimulation causes an increase in spinal cord Fos expression via capsaicin-insensitive primary afferent A␦ fibers, suggest that the most effective way to produce analgesia by peripheral nerve stimulation would be by high-frequency stimulation with an intensity strong enough to activate A␦ fibers.5,22 This concept is compatible with our results showing that activation of capsaicin-insensitive primary afferents (CIPAs) by peripheral chemical stimulation with diluted BV is most likely involved in BVAN. Because most A␦ afferents are insensitive to capsaicin,25 it is likely that BV is producing its antinociceptive effect by activation of A␦ fibers at the site of injection.

Role of Capsaicin-Insensitive Afferents in the Central Neuronal Mechanisms Underlying BV-Induced Antinociception We have recently demonstrated that peripheral BV injection increases Fos expression in rat brainstem catecholaminergic neurons including many neurons in the LC.19 We have further shown that the activation of spinal ␣2-adrenoceptors, but not opioid receptors, is critically involved in the BV-induced antinociceptive and antihyperalgesic effects observed in rodent models of visceral pain, inflammatory pain, and neuropathic pain.16,17,30 As an extension of this work, the present study demonstrated that chemical stimulation

References 1. Carlton SM, Zhou S, Kraemer B, Coggeshall RE: A role for peripheral somatostatin receptors in counter-irritation-induced analgesia. Neuroscience 120:499-508, 2003 2. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389:816-824, 1997 3. Chen J, Luo C, Li HL: The contribution of spinal neuronal changes to development of prolonged, tonic nociceptive responses of the cat induced by subcutaneous bee venom injection. Eur J Pain 2:359-376, 1998

511 of the Zusanli acupoint by BV leads to activation of peripheral CIPAs fibers, which in turn evokes catecholaminergic neuronal activation in the LC region. Because the coeruleospinal pathway plays an important role in the descending pain inhibitory system,38 activation of the LC by BV stimulation of CIPAs likely plays a key role in BV’s antinociceptive effects on formalin-induced nociceptive behaviors. The fact that RTX 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 noradrenergic system via stimulation of peripheral CIPAs. This is further supported by the finding that intrathecal pretreatment with the ␣2-adrenoceptor antagonist IDA abolished BV’s antinociceptive effect on formalininduced pain behavior, whereas RTX pretreatment failed to alter IDA’s inhibitory effect on BVAN. Collectively these data indicate that BV injection into the Zusanli acupoint stimulates peripheral CIPAs, which in turn activate catecholaminergic neurons in the LC. The LC then stimulates spinal cord ␣2-adrenoceptors via descending noradrenergic pathways, and this leads to BV’s 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 antinociceptive effect in the second phase of the formalin test that involves spinal neuronal transmission without detectable nociceptive behavior, and (2) peripheral CIPAs are primarily involved in activating central catecholaminergic pathways associated with BV’s antinociceptive effect.

7. De Koninck Y, Henry JL: Prolonged GABAA-mediated inhibition following single hair afferent input to single spinal dorsal horn neurones in cats. J Physiol 476:89-100, 1994 8. Doyle CA, Hunt SP: Substance P receptor (neurokinin-1)expressing neurons in lamina I of the spinal cord encode for the intensity of noxious stimulation: a c-Fos study in rat. Neuroscience 89:17-28, 1999 9. Fukura M, Kashima K, Maeda S, Shiba R: Changes in bite force and muscle forces in the upper extremities after counter irritation. Cranio 22:45-49, 2004 10. Hylden JL, Wilcox GL: Intrathecal morphine in mice: A new technique. Eur J Pharmacol 67:313-316, 1980

4. Chen J, Chen HS: Pivotal role of capsaicin-sensitive primary afferents in development of both heat and mechanical hyperalgesia induced by intraplantar bee venom injection. Pain 91:367-376, 2001

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

5. Chung JM, Lee KH, Hori Y, Endo K, Willis WD: Factors influencing peripheral nerve stimulation produced inhibition of primate spinothalamic tract cells. Pain 19:277-293, 1984

12. Jinks SL, Simons CT, Dessirier JM, Carstens MI, Antognini JF, Carstens E: C-fos induction in rat superficial dorsal horn following cutaneous application of noxious chemical or mechanical stimuli. Exp Brain Res 145:261-269, 2002

6. Coggeshall RE: Fos, nociception and the dorsal horn. Prog Neurobiol 77:299-352, 2005

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

512 14. Kim HW, Kwon YB, Ham TW, Roh DH, Yoon SY, Lee HJ, Han HJ, Yang IS, Beitz AJ, Lee JH: Acupoint stimulation using bee venom attenuates formalin-induced pain behavior and spinal cord fos expression in rats. J Vet Med Sci 65:349-355, 2003 15. Kim HW, Kwon YB, Ham TW, Roh DH, Yoon SY, Kang SY, Yang IS, Han HJ, Lee HJ, Beitz AJ, Lee JH: General pharmacological profiles of bee venom and its water soluble fractions in rodent models. J Vet Sci 5:309-318, 2004 16. Kim HW, Kwon YB, Han HJ, Yang IS, Beitz AJ, Lee JH: Antinociceptive mechanisms associated with diluted bee venom acupuncture (apipuncture) in the rat formalin test: Involvement of descending adrenergic and serotonergic pathways. Pharmacol Res 51:183-188, 2005 17. Kwon YB, Kang MS, Han HJ, Beitz AJ, Lee JH: Visceral antinociception produced by bee venom stimulation of the Zhongwan acupuncture point in mice: Role of alpha(2) adrenoceptors. Neurosci Lett 308:133-137, 2001 18. Kwon YB, Lee JD, Lee HJ, Han HJ, Mar WC, Kang SK, Beitz AJ, Lee JH: Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses. Pain 90:271-280, 2001 19. Kwon YB, Han HJ, Beitz AJ, Lee JH: Bee venom acupoint stimulation increases Fos expression in catecholaminergic neurons in the rat brain. Mol Cells 17:329-333, 2004

Mechanism of Bee Venom–Induced Antinociception 29. Roby-Brami A, Bussel B, Willer JC, Le Bars D: An electrophysiological investigation into the pain-relieving effects of heterotopic nociceptive stimuli: Probable involvement of a supraspinal loop. Brain 110:1497-1508, 1987 30. Roh DH, Kwon YB, Kim HW, Ham TW, Yoon SY, Kang SY, Han HJ, Lee HJ, Beitz AJ, Lee JH: Acupoint stimulation with diluted bee venom (apipuncture) alleviates thermal hyperalgesia in a rodent neuropathic pain model: involvement of spinal alpha 2-adrenoceptors. J Pain 5:297-303, 2004 31. Shibata M, Ohkubo T, Takahashi H, Inoki R: Modified formalin test: Characteristic biphasic pain response. Pain 38: 347-352, 1989 32. Szallasi A, Joo F, Blumberg PM: Duration of desensitization and ultrastructural changes in dorsal root ganglia in rats treated with resiniferatoxin, an ultrapotent capsaicin analog. Brain Res 503:68-72, 1989 33. Szolcsanyi J, Pinter E, Helyes Z, Oroszi G, Nemeth J: Systemic anti-inflammatory effect induced by counter-irritation through a local release of somatostatin from nociceptors. Br J Pharmacol 125:916-922, 1998 34. Takahashi Y, Chiba T, Kurokawa M, Aoki Y: Dermatomes and the central organization of dermatomes and body surface regions in the spinal cord dorsal horn in rats. J Comp Neurol 462:29-41, 2003

20. Lariviere WR, Melzack R: The bee venom test: Comparisons with the formalin test with injection of different venoms. Pain 84:111-112, 2000

35. Tender GC, Walbridge S, Olah Z, Karai L, Iadarola M, Oldfield EH, Lonser RR: Selective ablation of nociceptive neurons for elimination of hyperalgesia and neurogenic inflammation. J Neurosurg 102:522-525, 2005

21. Lee JH, Kwon YB, Han HJ, Mar WC, Lee HJ, Yang IS, Beitz AJ, Kang SK: Bee venom pretreatment has both an antinociceptive and anti-inflammatory effect on carrageenan-induced inflammation. J Vet Med Sci 63:251-259, 2001

36. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21:531-543, 1998

22. Lee KH, Chung JM, Willis WD Jr: Inhibition of primate spinothalamic tract cells by TENS. J Neurosurg 62:276-287, 1985

37. Torsney C, Meredith-Middleton J, Fitzgerald M: Neonatal capsaicin treatment prevents the normal postnatal withdrawal of A fibres from lamina II without affecting fos responses to innocuous peripheral stimulation. Brain Res Dev Brain Res 121:55-65, 2000

23. Luo C, Chen J, Li HL, Li JS: Spatial and temporal expression of c-Fos protein in the spinal cord of anesthetized rat induced by subcutaneous bee venom injection. Brain Res 806:175-185, 1998 24. Mansikka H, Lahdesmaki J, Scheinin M, Pertovaara A: Alpha2A adrenoceptors contribute to feedback inhibition of capsaicin-induced hyperalgesia. Anesthesiology 101:185190, 2004 25. Nagy JI, van der Kooy D: Effects of neonatal capsaicin treatment on nociceptive thresholds in the rat. J Neurosci 3:1145-1150, 1983 26. Nakatsuka T, Furue H, Yoshimura M, Gu JG: Activation of central terminal vanilloid receptor-1 receptors and alpha beta-methylene-ATP-sensitive P2X receptors reveals a converged synaptic activity onto the deep dorsal horn neurons of the spinal cord. J Neurosci 22:1228-1237, 2002

38. Tsuruoka M, Maeda M, Nagasawa I, Inoue T: Spinal pathways mediating coeruleospinal antinociception in the rat. Neurosci Lett 362:236-239, 2004 39. Uchida Y, Nishigori A, Takeda D, Ohshiro M, Ueda Y, Ohshima M, Kashiba H: Electroacupuncture induces the expression of Fos in rat dorsal horn via capsaicin-insensitive afferents. Brain Res 978:136-140, 2003 40. Williams S, Evan GI, Hunt SP: Changing patterns of c-fos induction in spinal neurons following thermal cutaneous stimulation in the rat. Neuroscience 36:73-81, 1990 41. Wismer CT, Faltynek CR, Jarvis MF, McGaraughty S: Distinct neurochemical mechanisms are activated following administration of different P2X receptor agonists into the hindpaw of a rat. Brain Res 965:187-193, 2003

27. Pan HL, Khan GM, Alloway KD, Chen SR: Resiniferatoxin induces paradoxical changes in thermal and mechanical sensitivities in rats: Mechanism of action. J Neurosci 23:29112919, 2003

42. Yajiri Y, Yoshimura M, Okamoto M, Takahashi H, Higashi H: A novel slow excitatory postsynaptic current in substantia gelatinosa neurons of the rat spinal cord in vitro. Neuroscience 76:673-688, 1997

28. Pericic D: Swim stress inhibits 5-HT2A receptor-mediated head twitch behaviour in mice. Psychopharmacology 167:373-379, 2003

43. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109-110, 1983