Persistent Spontaneous Nociception Research Articles

Schwann cell-derived CXCL2 contributes to cancer pain by modulating macrophage infiltration in a mouse breast cancer model

Pain is one of the most severe complications affecting the quality of life of cancer patients. Although substantial progress has been made in the diagnosis and treatment of cancer, the neurobiological mechanism of cancer pain is still unclear. In the present study, we identified the critical role of CXC chemokine 2 (CXCL2), released by Schwann cells after being activated by cancer cells, in maintaining cancer-induced macrophage infiltration and the resulting mechanical hypersensitivity and persistent spontaneous nociception. In vitro, Schwann cells cocultured with breast cancer cells exhibited a significant increase in CXCL2 expression; in addition, conditioned medium from Schwann cells activated by breast cancer cells had a similar effect to recombinant CXCL2 in terms of inducing macrophage migration. Targeting CXCL2 signaling by both CXC chemokine receptor 2 (CXCR2) antagonist pharmacological blockade and anti-CXCL2 mAb immunological blockade robustly prevented conditioned medium-induced macrophage migration. In vivo, both application of recombinant CXCL2 and perineural breast cancer cell implantation resulted in mechanical hypersensitivity and persistent spontaneous nociception in mice, along with increased macrophage infiltration into the sciatic nerves. Similar to the in vitro results, inhibition of CXCL2/CXCR2 signaling or conditional knockdown of CXCL2 in sciatic nerve Schwann cells effectively attenuated breast cancer cell-induced mechanical hypersensitivity, persistent spontaneous nociception, and macrophage recruitment in the sciatic nerve. Mechanistically, we found that redox effector factor-1 (Ref-1) secreted by breast cancer cells activated hypoxia inducible factor-1α (HIF-1α) expression and inhibited reactive oxygen species (ROS) production in Schwann cells, ultimately inducing CXCL2 expression in Schwann cells. In brief, the present study expands new insights into cancer pain mechanisms from promising animal models to provide new strategies for the control of cancer pain.

Activation of peripheral group III metabotropic glutamate receptors suppressed formalin-induced nociception.

Intraplantar injection of formalin produces persistent spontaneous nociception and hyperalgesia. The underlying mechanism, however, remains unclear. The present study was, therefore, designed to determine the roles of peripheral group III metabotropic glutamate receptors (mGluRs) in formalin-evoked spontaneous nociception. Pre-treatment with intraplantar injections of L-serine-O-phosphate (L-SOP), a group III mGluRs agonist, significantly inhibited formalin-induced nociceptive behaviours and decreased Fos production in the spinal dorsal horn. The inhibitory effects of L-SOP were abolished completely by pre-treatment with the group III mGluR antagonist (RS)-a-methylserine-O-phosphate (M-SOP). These data suggest that the activation of group III mGluRs in the periphery may play a differential role in formalin-induced nociception. In addition, L-SOP decreased the formalin-induced upregulation of tumour necrosis factor-α (TNF-α) as well as interleukine-1β (IL-1β) expression in the spinal cord, suggesting that activation of peripheral group III mGluRs reduces formalin-induced nociception through inhibition of the pro-inflammatory cytokines in the spinal cord. Therefore, the agonists acting peripheral group III mGluRs possess therapeutic effectiveness in chronic pain.

Antinociceptive effects of systemic tanshinone IIA on visceral and somatic persistent nociception and pain hypersensitivity in rats

Previous studies showed that tanshinone IIA (TIIA), an important lipophilic component of Danshen, has been well-established to exhibit various neuroprotective actions in the nervous system. Although we previously reported that TIIA had a significant anti-nociceptive effect in complete Freund's adjuvant (CFA)-induced pain, it is surprisingly noted that few pharmacological studies have been carried out to explore the possible analgesic action of TIIA in animals and the appropriate indications for treatment of clinical pain remain unclear. Therefore, in the present study, by using both somatic and visceral pain models, the antinociceptive and antihyperalgesic effects of TIIA were evaluated by intraperitoneal administration in rats. In the bee venom (BV) test, when compared with saline controls, systemic pre- and post-treatment with TIIA resulted in an apparent antinociception against persistent spontaneous nociception (PSN) and primary heat and mechanical hypersensitivity, while for the mirror-image heat hypersensitivity, only pre-treatment was effective. Moreover, in the formalin test, the antinociception of TIIA was significant for both phases 1 and 2 in the pretreatment groups, but only effective for phase 2 in the post-treatment group. In the acetic acid writhing test, the number of writhes was effectively blocked by both pre- and post-treatment of TIIA. Taken together, these results provide a new line of evidence showing that TIIA is also able to produce analgesia against various ‘phenotypes’ of nociception and hypersensitivity.

Contribution of the spinal microglia to bee venom-induced inflammatory pain in conscious rats

It is well known that spinal glia plays a key role in the pathogenesis of pain. The present study was designed to determine the roles of spinal microglia in bee venom-induced persistent spontaneous nociception (PSN), mechanical hyperalgesia and inflammation. We determined the effects of microglia inhibitor minocycline on BV-induced PSN, mechanical hyperalgesia and inflammatory swelling. Pre-treatment with intrathecal administration of minocyline at different doses significantly inhibited BV-induced PSN and mechanical hyperalgesia, but had no effect on BV-induced inflammatory swelling. These data suggest that the activation of spinal microglia may play a key role in BV-induced nociception, but not inflammation.

Characterization of nociceptive responses to bee venom-induced inflammation in neonatal rats

To assess developmental characteristics of nociceptive responses induced by bee venom (BV) injection in neonatal rats, we exposed pups to intra-plantar injection of various BV concentrations given at different time points between postnatal day 1 and day 28 (P1–P28). Persistent spontaneous nociception (PSN) as well as thermal and mechanical nociceptive response was compared before and after a BV injection. There were distinct age-related changes in the baseline paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWMT) when examined on P1, P4, P7, P14, P21, and P28. The lowest and highest baseline PWTL was shown on P1 and P7, respectively, and PWTL was unchanged from P7 to P28. In contrast, the baseline PWMT remained low before P21 but increased dramatically afterward. Neonatal rats receiving intra-plantar BV injection showed a time-dependent change in nociceptive responses, including (1) a dose-related increase in PSN from P1 to P28; (2) a non-specific decrease (indistinguishable between saline and BV injection) in PWTL and PWMT up to P14 and P21, respectively; and (3) a specific decrease (in response to BV injection only) in PWTL and PWMT after P14 and P21, respectively. These findings indicate that characteristic changes in the baseline and BV-induced nociceptive response are both time-dependent and modality-specific in neonatal rats. The data reveal a critical postnatal period during which nociceptive stimulation could have a significant influence on nociceptive behavior in adult rats and suggest that preclinical models of neonatal nociception should be evaluated according to different postnatal time points.

Effects of a non-selective TRPC channel blocker, SKF-96365, on melittininduced spontaneous persistent nociception and inflammatory pain hypersensitivity

Melittin is the main peptide in bee venom and causes both persistent spontaneous nociception and pain hypersensitivity. Our recent studies indicated that both transient receptor potential (TRP) vanilloid receptor 1 (TRPV1) and canonical TRPs (TRPCs) are involved in mediating the melittin-induced activation of different subpopulations of primary nociceptive cells. Here, we further determined whether TRPC channels are involved in melittin-induced inflammatory nociceptive responses in behavioral assays. The anti-nociceptive and anti-hyperalgesic effects of localized peripheral administration of three doses of the non-selective TRPC antagonist, SKF-96365 (1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl}-1H-imidazole hydrochloride), were evaluated in melittin tests. Pain-related behaviors were rated by counting the number of paw flinches, and measuring paw withdrawal thermal latency (s) and paw withdrawl mechanical threshold (g), over a 1-h time-course. Localized peripheral SKF-96365 given before melittin prevented, and given after melittin significantly suppressed, the melittin-evoked persistent spontaneous nociception. Pre-blockade and post-suppression of activation of primary nociceptive activity resulted in decreased hypersensitivity to both thermal and mechanical stimuli applied to the primary injury site of the ipsilateral hindpaw, despite dose-effect differences between thermal and mechanical hyperalgesia. However, local administration of SKF-96365 into the contralateral hindpaw had no significant effect on any pain-associated behaviors. In addition, SKF-96365 had no effect on baseline threshold for either thermal or mechanical sensitivity under normal conditions. Besides TRPV1, SKF-96365-sensitive TRPC channels might also be involved in the pathophysiological processing of melittin-induced inflammatory pain and hypersensitivity. Therapeutically, SKF-96365 is equally effective in preventing primary thermal and mechanical hyperalgesia as well as persistent spontaneous nociception. However, this drug is likely to be more effective in the relief of thermal hyperalgesia than mechanical hyperalgesia when applied 5 min after establishment of primary afferent activation.

Suppressive Effects of Intrathecal Paeoniflorin on Bee Venom-Induced Pain-Related Behaviors and Spinal Neuronal Activation

Recently, paeoniflorin (PF) administered systemically was found to have analgesic effects against inflammatory pain and hypersensitivity in a naloxone-reversible manner. In the present study, we adopted intrathecal administration to evaluate whether PF has direct antinociceptive actions at the spinal level. Pain-related behaviors and spinal c-Fos expression were induced by subcutaneous injection of bee venom (BV) into one hind paw of a rat. Intrathecal pretreatment of PF resulted in an inhibition of the BV-induced persistent spontaneous nociception and partially suppressed the occurrence of both thermal and mechanical hypersensitivity. Moreover, the PF-produced antinociception was completely reversed by naloxone. We further evaluated the intrathecal effects of the drug on the BV-induced c-Fos expression. The result showed that intrathecal PF preconditioning was effective to suppress spinal c-Fos expression in both superficial (lamina I–II) and deep (lamina IV–VI) layers of the L_4–5 dorsal spine. This result showed that PF has a direct pharmacological action in the spinal cord dorsal horn via activation of opioid receptors.

Effects of SKF-96365, a TRPC inhibitor, on melittin-induced inward current and intracellular Ca2+ rise in primary sensory cells

Melittin (MEL) is a major component of bee venom and can produce both persistent spontaneous nociception and pain hypersensitivity when injected subcutaneously in the periphery. The present study aimed to examine the roles of transient receptor potential canonical (TRPC) channels in mediation of MEL-induced activation of primary nociceptive cells. Whole-cell patch-clamp and laser scanning confocal calcium detection were used to evaluate the effects of SKF-96365, a TRPC inhibitor, applied on the acutely isolated dorsal root ganglion (DRG) cells of rat, on MEL-induced increase in intracellular calcium concentration ([Ca(2+)](i)) and inward current. Under voltage-clamp mode, 43.9% (40/91) DRG cells were evoked to give rise to the inward current by 2 μmol/L MEL, which could be significantly suppressed by 3 doses of SKF-96365 (1, 5 and 10 μmol/L) in a dose-dependent manner. Of the other 210 cells, 67.6% responded to MEL with an intracellular Ca(2+) rise, as revealed by confocal calcium imaging. Of these MEL-sensitive cells, 46.5% (66/142) were suppressed by the highest dose of SKF-96365. MEL-induced activation of small to medium-sized DRG cells can be suppressed by SKF-96365, suggesting the involvement of TRPC channels in the mediation of MEL-induced activation of primary nociceptive cells.

The anti-nociceptive effect and the possible mechanism of acupoint stimulation caused by chemical irritants in the bee venom pain model

Many studies have demonstrated the anti-nociceptive and anti-inflammatory effects of injecting bee venom (BV) into the Zusanli (ZSL) acupoint in rats. The present study was designed to determine whether the injection of other chemical irritants, such as formalin and complete Freund's adjuvant (CFA), into the ZSL acupoint can produce anti-nociceptive and anti-inflammatory effects in the BV pain model and to determine the possible mechanisms underlying these effects. First, the effects of injecting BV, formalin, CFA, or saline into the ZSL acupoint on intraplantar BV-induced persistent spontaneous pain, mechanical hyperalgesia, and inflammatory swelling of the injected paw were observed. BV, formalin, CFA, and saline injection into the ZSL acupoint significantly inhibited intraplantar BV-induced persistent spontaneous nociception (PSN) and mechanical hyperalgesia but had no effect on intraplantar BV-induced inflammatory swelling. Next, the effects of pretreatment with naloxone (5 mg/kg, ip) or injection of 0.15% capsaicin into the ZSL acupoint on the anti-nociceptive effect of BV acupuncture (BVA) were observed. Pretreatment with naloxone had no effect on the BVA-induced anti-nociceptive effect, intraplantar BV-induced PSN, and mechanical hyperalgesia. Pretreatment with capsaicin produced partial blockage of the BVA-induced anti-nociceptive effect on PSN, but it had no effect on BVA-induced anti-nociception of mechanical hyperalgesia. These results suggest that (1) chemical irritant acupuncture produces the anti-nociceptive effect but not the anti-inflammatory effect in the BV pain model, and (2) chemical irritant acupuncture-induced analgesia is a common mechanism that is not specific to BV acupuncture. Our results also suggest that the BVA-induced anti-nociceptive mechanism is partially mediated by capsaicin-sensitive primary afferent fibers but not by endogenous mu opioid receptors in the BV pain model.

Differential Roles of Peripheral Metabotropic Glutamate Receptors in Bee Venom-Induced Nociception and Inflammation in Conscious Rats

Intraplantar injection of bee venom (BV) produces persistent spontaneous nociception (PSN), hyperalgesia, and inflammatory swelling of the injected paw. The present study was designed to determine the roles of peripheral metabotropic glutamate receptors (mGluRs) in BV-induced nociception and inflammation. We determined the effects of the group I mGluR antagonist AIDA, the group II mGluR agonist ADPC, and the group III mGluR agonist L-AP4 on BV-induced PSN, mechanical hyperalgesia, and inflammatory swelling. Pretreatment with intraplantar injections of AIDA, ADPC or L-AP4 at different doses significantly inhibited BV-induced PSN over the 1-hour observational period. The inhibitory effects of ADPC and L-AP4 were completely abolished by pretreatment with the group II mGluR antagonist LY341495 and the group III mGluR antagonist MSOP, respectively. Pretreatment with ADPC prevented the BV-induced decrease in paw-withdrawal mechanical threshold (PWMT) in a dose-dependent manner, while pretreatment with AIDA or L-AP4 had no effect. The antihyperalgesic effect of ADPC was completely abolished by pretreatment with LY341495. Pretreatment with AIDA, ADPC or L-AP4 at different doses had no effect on the BV-induced increase in the paw volume (PV), a measurement of inflammatory swelling. All contralateral drug treatments at the highest doses had no effect on BV-induced PSN, decreases in PWMT or increases in PV, eliminating the possibility of drug-induced systemic effects. These data suggest that the activation of mGluRs in the periphery may play a differential role in BV-induced nociception and inflammation. The present study demonstrated that the intraplantar injection of antagonists or agonists of different mGluRs produced differential effects on bee venom-induced persistent spontaneous nociception and mechanical hyperalgesia. However, no effects on inflammation were observed, suggesting that mGluRs in the periphery have differential roles. Thus, therapies specifically targeting metabotropic glutamate receptors may improve the treatment of patients with persistent spontaneous nociception and hyperalgesia.

Differential Roles of Peripheral Mitogen-Activated Protein Kinase Signal Transduction Pathways in Bee Venom–Induced Nociception and Inflammation in Conscious Rats

Intraplantar injection of bee venom (BV) produces persistent spontaneous nociception (PSN) and hyperalgesia, as well as obvious inflammatory swelling, in the paws of injected rats. The present study was designed to determine the peripheral roles of mitogen-activated protein kinase (MAPK) signal transduction pathways in BV-induced nociception and inflammation. We examined the effect of intraplantar injection of an ERK1/2 inhibitor, PD98059, and a p38 inhibitor, SB202190, on BV-induced PSN, mechanical hyperalgesia, and inflammatory swelling. We found that (1) pretreatment with SB202190 (0.1 to 10 microg) had no effect on BV-induced PSN, whereas pretreatment with PD98059 (0.1 to 100 microg) produced a significant and dose-dependent inhibition of BV-induced PSN; (2) pretreatment with PD98059 (0.1 to 100 microg) had no effect on BV-induced decreases in paw withdrawal mechanical threshold (PWMT), while pretreatment with SB202190 (0.1 to 10 microg) produced an obvious prevention of the BV-induced decrease in PWMT; and (3) pretreatment with PD98059 (0.1 to 100 microg) had no effect on BV-induced increase in paw volume (PV), whereas pretreatment with SB202190 (0.1 to 10 microg) produced a dose-related inhibition of BV-induced increases in PV. No contralateral drug treatments, even at the highest dose, had any effect on BV-induced PSN, PWMT or PV, ruling out the systemic effect of these drugs. These results suggest that peripheral MAPK signal transduction pathways may play differential roles in bee venom-induced nociception and inflammation. Targeting specific peripheral MAPKs might prove effective in the treatment of persistent pain and inflammation. The present article showed that intraplantar injection of different MAPK inhibitors produced differential effects on bee venom-induced nociception and inflammation, suggesting that the peripheral MAPK signal transduction pathways have differential roles. Targeting specific peripheral MAPKs might prove effective in the treatment of persistent pain and inflammation.

Roles of Peripheral P2X and P2Y Receptors in the Development of Melittin-Induced Nociception and Hypersensitivity

A recent report from our laboratory shows that subcutaneous (s.c.) injection of melittin could induce persistent spontaneous nociception (PSN) and primary thermal or mechanical hyperalgesia. However, the exact peripheral mechanisms underlying melittin-induced multiple pain-related behaviors remain unclear. In this study, behavioral tests combined with pharmacological manipulations were used to explore potential roles of local P2X and P2Y receptors in melittin-induced inflammatory pain and hyperalgesia. Post-treatment of the primary injury site with s.c. injection of A-317491 (a potent P2X(3)/P2X(2/3) receptor antagonist) and Reactive Blue 2 (a potent P2Y receptor antagonist) could significantly suppress the development of melittin-evoked PSN and hypersensitivity (thermal and mechanical). Our control experiments demonstrated that local administration of either antagonist into the contralateral hindpaw produced no significant effect on any kind of pain-associated behaviors. Taken together, these data indicate that activation of P2X and P2Y receptors might be essential to the maintenance of melittin-induced primary thermal and mechanical hyperalgesia as well as on-going pain.

Roles of peripheral mitogen-activated protein kinases in melittin-induced nociception and hyperalgesia

Recently, we have reported that melittin, a major toxic peptide of the whole bee venom, plays a central role in production of local inflammation, nociception and hyperalgesia following the experimental honeybee’s sting. However, the exact peripheral mechanisms underlying melittin-induced multiple pain-related behaviors are still less characterized. In the present study, we sought to investigate the potential roles of peripheral mitogen-activated protein kinases (MAPKs) in melittin-induced nociception and hyperalgesia by pre- and post-administration of three MAPK inhibitors, namely U0126 (1 μg, 10 μg) for extracellular signal-regulated kinase (ERK), SP600125 (10 μg, 100 μg) for c-Jun N-terminal kinase (JNK) and SB239063 (10 μg, 100 μg) for p38 MAPK, into the local inflamed area of one hind paw of rats. Both pre- and post-treatment with three drugs significantly suppressed the occurrence and maintenance of melittin-evoked persistent spontaneous nociception (PSN) and primary heat hyperalgesia, with little antinociceptive effect on mechanical hyperalgesia. In vehicle-treated group, ipsilateral injection of melittin produced no impact on thermal and mechanical sensitivity of the other hind paw, suggesting no occurrence of contralateral heat and mechanical hyperalgesia in the melittin test. In addition, local administration of each inhibitor into the contralateral hind paw exerted no significant influence on either PSN or heat/mechanical hyperalgesia tested in the primary injured hind paw, excluding the systemically pharmacological effects of the three drugs. Furthermore, local administration of the three compounds in naïve animals, respectively, did not change the basal pain sensitivity to either thermal or mechanical stimuli, suggesting lack of peripherally functional roles of the three MAPK subfamily members in normal pain sensitivity under the physiological state. Taken together, we conclude that activation of peripheral MAPKs, including ERK, JNK and p38, might contribute to the induction and maintenance of persistent ongoing pain and primary heat hyperalgesia in the melittin test. However, they are not likely to be involved in the processing of melittin-induced primary mechanical hyperalgesia, implicating a mechanistic separation between mechanical and thermal hyperalgesia in the periphery.

Different roles of spinal p38 and c-Jun N-terminal kinase pathways in bee venom-induced multiple pain-related behaviors

Our previous studies have established the idea that different types of pain induced by subcutaneous bee venom (BV) injection might be mediated by different spinal signaling pathways. To further testify this hypothesis, the present investigation was designed to detect whether spinal p38 and c-Jun N-terminal kinase (JNK) pathways are equally or differentially involved in the development of persistent spontaneous nociception (PSN), primary heat and mechanical hyperalgesia, and mirror-image heat (MIH) hypersensitivity in the BV model, by evaluating the effects of intrathecal (i.t.) pre-administration of a p38 inhibitor SB239063 and a JNK inhibitor SP600125 in the conscious rat. The results showed that i.t. pre-treatment with either SB239063 or SP600125 caused a significant prevention of BV-induced persistent paw flinching reflex in a dose-related manner, with the former exhibiting much stronger inhibition than the latter. Moreover, the same doses of SB239063 and SP600125 also exhibited different suppressive actions on the induction of primary heat hyperalgesia and MIH hypersensitivity. That is, SP600125 produced a larger increase of thermal latency than SB239063 in the injected paw, whereas SB239063 mainly affected the value measured in the non-injected paw. Pre-treatment with neither SB239063 nor SP600125 had any effect on BV-evoked mechanical hyperalgesia. Taken together, these data suggest that activation of p38 in the spinal cord preferentially contributes to the development of PSN and MIH hypersensitivity under pathological state, while spinal JNK signaling pathways might play more important roles in inducing primary heat hyperalgesia.

Antinociceptive effects of systemic paeoniflorin on bee venom-induced various ‘phenotypes’ of nociception and hypersensitivity

Paeoniflorin (PF), one of the active chemical compounds identified from the root of Paeonia lactiflora Pall, has been well-established to exhibit various neuroprotective actions in the central nervous system (CNS) after long-term daily administration. In the present study, by using the bee venom (BV) model of nociception and hypersensitivity, antinociceptive effects of PF were evaluated by intraperitoneal administration in conscious rats. When compared with saline control, systemic pre- and post-treatment with PF resulted in an apparent antinociception against both persistent spontaneous nociception and primary heat hypersensitivity, while for the primary mechanical hypersensitivity only pre-treatment was effective. Moreover, pre- and early post-treatment with PF (5 min after BV injection) could successfully suppress the occurrence and maintenance of the mirror-image heat hypersensitivity, whereas late post-treatment (3 h after BV) did not exert any significant impact. In the Rota-Rod treadmill test, PF administration did not affect the motor coordinating performance of rats. Furthermore, systemic PF application produced no significant influence upon BV-induced paw edema and swelling. Finally, the PF-produced antinociception was likely to be mediated by endogenous opioid receptors because of its naloxone-reversibility. Taken together, these results provide a new line of evidence showing that PF, besides its well-established neuroprotective actions in the CNS, is also able to produce analgesia against various ‘phenotypes’ of nociception and hypersensitivity via opioid receptor mediation.

Roles of capsaicin-sensitive primary afferents in differential rat models of inflammatory pain: A systematic comparative study in conscious rats

To characterize the role of capsaicin-sensitive primary afferents in inflammatory pain, the effects of subcutaneous (s.c.) injection of 0.15% capsaicin on different chemical irritants-induced pathological nociception including persistent spontaneous nociception, primary thermal and mechanical hyperalgesia, and inflammatory response were systematically investigated in unanesthetized conscious rats. Four different animal models of inflammatory pain: the bee venom (BV) test, the formalin test, the carrageenan model, and the complete Freund’s adjuvant (CFA) model, were employed and compared. Local pre-treatment with capsaicin produced a significant inhibition on the s.c. BV and formalin induced long-lasting persistent spontaneous nociception. However, this capsaicin-induced inhibitory effect on spontaneous nociception in the BV test was only found within the late phase (tonic nociception; 11–60 min), but not the early phase (acute nociception; 0–10 min). A complete preventing effect of capsaicin on the decreased thermal paw withdrawal latency was found in the BV, carrageenan, and CFA models. Nevertheless, pre-treatment with capsaicin only produced complete blocking effects on the decreased mechanical paw withdrawal threshold in the BV and carrageenan models, but not in the CFA model. For inflammatory response, a significant inhibition of the BV-elicited paw swelling was found following capsaicin treatment. In marked contrast, capsaicin did not produce any effects on the paw inflammation during exposure to carrageenan, CFA, and formalin. These data suggest that capsaicin-sensitive primary afferents may play differential roles in the induction and development of pathological nociception in differential inflammatory pain models. In contrast to other chemical irritants, BV-induced long-term spontaneous nociception, facilitated nociceptive behavior, and inflammation are modulated by peripheral capsaicin-sensitive afferents.

Activation of spinal extracellular signaling-regulated kinases by intraplantar melittin injection

Intraplantar injection of melittin, a major toxic peptide of whole bee venom, has been proved to cause alteration in both behavioral and spinal neuronal responses in rats. To see whether extracellular signaling-regulated kinases (ERK) in the spinal cord dorsal horn are activated and involved in induction and maintenance of persistent ongoing nociception, pain hypersensitivity and inflammation, three doses of U0126 (1,4-diamino-2,3-dicyano-1, 4-bis-[ o-aminophenylmercapto]butadiene), a widely used specific MAP kinase kinase (MEK) inhibitor, were administered through chronic intrathecal catheterization prior to or after intraplantar injection of melittin. We found that: (1) the induction of melittin-induced persistent spontaneous nociception (PSN), mechanical and heat hypersensitivity could be suppressed by U0126 in a dose-related manner; (2) specific inhibition of ERK pathway suppressed the maintenance of melittin-induced PSN and heat hypersensitivity, while established mechanical hypersensitivity could not be reversed; and (3) intrathecal administration of U0126 had no effects on peripheral inflammation induced by melittin. This result suggests that spinal ERK pathway might be a common factor involved in inducing and maintaining pathophysiological processes of ongoing pain and heat hyperalgesia, while the role of ERK pathway in generation of the mechanical hypersensitivity is not consistent and remains to be further clarified.

Altered pain-related behaviors and spinal neuronal responses produced by s.c. injection of melittin in rats

Recently, we have reported that following s.c. injection of a solution containing the whole bee-venom (BV; Apis mellifera), into one hind paw of a rat, the experimentally produced honeybee's sting, the animal shows altered pain-related behaviors and inflammation relevant to pathological pain state. To see whether melittin, the major (over 50%) toxic component of the BV, is responsible for the above abnormal pain behavioral changes, the present study was designed to investigate the effects of s.c. melittin on either nociceptive behaviors in conscious rats or spinal dorsal horn neuronal responses in anesthetized rats. In the behavioral surveys, s.c. injection of three doses of both melittin (5, 25 and 50 μg) and BV (10, 50 and 100 μg) into the posterior surface of one hind paw of rats produced an immediate tonic nociceptive response displaying as persistent spontaneous paw flinching reflex. Similar to the BV test, the melittin response was also monophasic and dose-dependent in terms of both intensity and time course. As an accompanied consequence, both heat and mechanical hypersensitivity (hyperalgesia and allodynia) and inflammatory responses (paw swelling and plasma extravasation) were induced by s.c. melittin injections. In the electrophysiological recordings, s.c. injection of the same three doses of melittin into the cutaneous receptive field produced an immediate, dose-dependent increase in spontaneous spike discharges of spinal dorsal horn wide-dynamic-range (WDR) neurons which are believed to be responsible for the spinally-organized nociceptive flexion reflex. The melittin-induced ongoing spike responses are similar to the behavioral flinching reflex in terms of both duration and frequency. Furthermore, the responsiveness of the WDR neurons to both heat (42 °C, 45 °C, 47 °C and 49 °C) and mechanical (brush, pressure and pinch) stimuli was significantly enhanced by s.c. injection of melittin shown as a leftward shift of the stimulus-response functional curves. Taken together, the present results suggest that melittin, the major toxin of the whole BV, is likely to be responsible for production of the long-term spinal neuronal changes as well as persistent spontaneous nociception, heat/mechanical hypersensitivity and inflammatory responses that are produced by experimental honeybee's sting.

Supraspinal contribution to development of both tonic nociception and referred mirror hyperalgesia: a comparative study between formalin test and bee venom test in the rat.

The roles of descending facilitatory pathway from the rostral medial medulla (RMM) in development of persistent spontaneous nociception and hyperalgesia were evaluated in the bee venom (BV) test and the formalin test. Bilateral lesions of the RMM with ibotenic acid, a soma-selective neurotoxin, were performed to study their effects on the spontaneous pain-related behaviors and hyperalgesia, which were determined by counting the number of flinching reflex per 5 min (1 h) and by measuring paw withdrawal thermal latency (PWTL) and mechanical threshold (PWMT) to radiant heat and von-Frey filaments to both hind paws in conscious rats, respectively. 1) Bilateral lesions of the RMM produced a similarly significant inhibition of persistent spontaneous flinching reflexes in the BV test and the formalin test; however, the inhibitory effect occurred in the late 50 min (11-60 min), but not the first 10 min (0-10 min) following intraplantar injection of either BV or formalin. 2) Bilateral lesions of the RMM prevented the development of the BV-induced referred mirror heat hyperalgesia occurred in the noninjected paw, but had no effect on the primary heat and mechanical hyperalgesia occurred in the injected paw. The present results provide a new line of behavioral evidence that tonic activation of descending facilitatory pathway contributes to the establishment of 1) the BV and formalin-induced persistent spontaneous nociception; and 2) the BV-induced referred mirror heat hyperalgesia and the central sensitization, but not the primary heat and mechanical hyperalgesia.

Differential Roles of Spinal Protein Kinases C and A in Development of Primary Heat and Mechanical Hypersensitivity Induced by Subcutaneous Bee Venom Chemical Injury in the Rat

It has been demonstrated that subcutaneous injection of bee venom (BV) can produce different types of pain and hypersensitivity including persistent spontaneous nociception (PSN), primary heat and mechanical hypersensitivity (hyperalgesia) and mirror-image heat (MIH) hypersensitivity in an individual animal, and the changes of spinal neurons are likely to be responsible for the production of these pain-related behaviors. In this study, we examined the roles of spinal protein kinase C (PKC) and protein kinase A (PKA) in the BV-induced different types of pain and hypersensitivity in conscious rats. We found that: (1) BV-induced primary heat hypersensitivity could be blocked by intrathecal pre- or posttreatment with a PKC inhibitor, chelerythrine chloride (CH), while a PKA inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89), had no effect. (2) BV-induced primary mechanical hypersensitivity could be blocked by pre- or posttreatment with H89, whereas CH had no effect. (3) Both pre- and posttreatment with H89 produced suppressive effects on both induction and maintenance of the BV-induced PSN and MIH hypersensitivity. Based on the present findings, we proposed that spinal PKC might be activated during the central processes of primary heat hypersensitivity, while spinal PKA is likely to be involved in primary mechanical hypersensitivity induced by subcutaneous BV chemical injury. Taken together with our previous report however, spinal PKC and PKA are likely to be simultaneously involved in the central processes of both PSN and MIH hypersensitivity.