Tomoyuki Kawamata

Formalin-induced release of excitatory amino acids in the skin of the rat hindpaw

Application of glutamate to skin evokes pain-related behaviors [S.M. Carlton, G.L. Hargett, R.E. Coggeshall, Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin, Neurosci. Lett., 197 (1995) 25-28; D.L. Jackson, C.B. Graff, J.D. Richardson, K.M. Hargreaves, Glutamate participates in the peripheral modulation of thermal hyperalgesia in rats, Eur. J. Pharmacol., 284 (1995) 321-325.] and peripherally-administered glutamate antagonists can prevent the nociception produced by inflammation [E.M. Davidson, R.E. Coggeshall, S.M. Carlton, Peripheral NMDA and non-NMDA glutamate receptors contribute to nociceptive behaviors in the rat formalin test, NeuroReport, 8 (1997) 941-946; Jackson et al., 1995.] In this study, the concentrations of glutamate and aspartate in the plantar of the rat hindpaws were measured before and after the subcutaneous administration of formalin. Increases in glutamate and aspartate concentrations were observed on the ipsilateral side, but not on the contralateral side, to the injection. This shows that nociception and inflammation caused by formalin injection induces the release of peripheral glutamate and aspartate, which would contribute to nociception and inflammatory pain.

Different Mechanisms of Development and Maintenance of Experimental Incision-induced Hyperalgesia in Human Skin

BACKGROUND To determine the mechanisms of postoperative pain, the effects of local anesthesia on development and maintenance of surgical incision-induced hyperalgesia were evaluated in a crossover, double-blinded, placebo-controlled human study using 17 subjects. METHODS An experimental 4-mm-long incision through skin, fascia, and muscle was made in the volar forearm of each subject. In experiment 1, 1% lidocaine or saline in a volume of 0.2 ml was subcutaneously injected into the incision site pretraumatically and posttraumatically. In experiment 2, a 5-cm-long strip of skin was subcutaneously injected with 0.2 ml of 1% lidocaine near the incision site pretraumatically and posttraumatically. Flare, spontaneous pain, and primary and secondary hyperalgesia to punctate mechanical stimuli were assessed after the incision had been made. RESULTS Pretraumatic lidocaine injection prevented the occurrence of spontaneous pain and development of flare formation that was found surrounding the incision site immediately (1 min) after the incision had been made. The lidocaine suppressed primary hyperalgesia more effectively than did posttraumatic block, but only for the first 4 h after the incision. The preincision block prevented development of secondary hyperalgesia, whereas posttraumatic block did not significantly affect the fully developed secondary hyperalgesia. The area of flare formation and the area of secondary hyperalgesia did not extend over the strip of the skin that had been pretraumatically anesthetized, whereas the posttraumatic block did not significantly reduce the area of fully developed secondary hyperalgesia. CONCLUSIONS Pretraumatic injection of lidocaine reduces primary hyperalgesia more effectively than does posttraumatic injection, but only for a short period after incision. The spread of secondary hyperalgesia is mediated peripheral nerve fibers, but when secondary hyperalgesia has fully developed, it becomes less dependent on or even independent of peripheral neural activity originating from the injured site.

Peripheral nitric oxide in carrageenan-induced inflammation

Recent studies have suggested that nitric oxide (NO) peripherally produced by different nitric oxide synthase (NOS) isoforms contributes to edema formation and development of hyperalgesia. The present study was designed to examine the effects of NOS isoforms on NO release in carrageenan-induced inflammation at various time points. A microdialysis probe was implanted subcutaneously into the glabrous skin of hindpaws of Sprague-Dawley rats under pentobarbital anesthesia. After sample collection to obtain the basal level of the total amount of nitrite and nitrate (NO2-/NO3-), modified Ringer solution, a non-selective NOS inhibitor, NG monomethyl-L-arginine acetate (L-NMMA), or an iNOS inhibitor, aminoguanidine hemisulfate (AG) was perfused through the microdialysis probe. 2 mg of carrageenan was injected into the plantar surface of the probe-implanted hindpaw. Carrageenan was also injected in rats that had undergone sciatic nerve sectioning. Carrageenan significantly increased the dialysate concentrations of NO2-/NO3- for more than 8 h. L-NMMA suppressed the carrageenan-induced increase in NO2-/NO3- concentration. Although AG did not suppress the increase in NO2-/NO3- for the first 2 h after carrageenan injection, significant suppression of the increase in NO2-/NO3- was observed from 2.5 h after carrageenan injection. In the rats in which the sciatic nerves had been denervated, the increases in concentrations of NO2-/NO3- were completely suppressed up to 3 h and partially suppressed 4.5-8 h after carrageenan injection. The results of the current study show that carrageenan induces peripheral release of NO, the production of which is mediated by nNOS in the early phase and by both nNOS and iNOS in the late phase of carrageenan-induced inflammation.

Bone cancer increases transient receptor potential vanilloid subfamily 1 expression within distinct subpopulations of dorsal root ganglion neurons.

Bone cancer pain has a strong impact on the quality of life of patients but is difficult to treat. Therefore, the mechanisms of bone cancer pain require elucidation for the purpose of development of new therapeutics. A recent study showed that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) was involved in bone cancer pain. In this study, we re-evaluated the analgesic effects of pharmacological blockade of TRPV1 using the potent TRPV1 antagonist 5-iodoresiniferatoxin (I-RTX) and examined whether bone cancer can change TRPV1 expression and distribution in the primary sensory neurons in a mouse model of bone cancer pain. Implantation of osteosarcoma into the femur induced ongoing and movement-evoked bone cancer-related pain behaviors. These behaviors were significantly reduced by i.p. administration of I-RTX, compared with vehicle. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that TRPV1 level was significantly increased in dorsal root ganglions (DRGs) ipsilateral to sarcoma implantation. Immunohistochemical analysis showed that implantation of osteosarcoma induced not only an increase in the percentage of TRPV1-positive neurons among DRG neurons (24.3+/-1.3% in sham mice and 31.2+/-1.3% in mice with osteosarcoma implantation, P<0.05) but also an overall shift in the distribution of area of profiles to the right. Colocalization study showed that the percentages of colocalization of TRPV1 with neurofilament 200 kD (NF200) and calcitonin gene-related peptide (CGRP) but not isolectin B4 (IB4) among DRG neurons in mice with osteosarcoma implantation were increased compared with those in sham mice (from 0.8+/-0.1% to 2.1+/-0.3% for TRPV1 and NF200 and from 21.1+/-1.3% to 26.5+/-0.2% for TRPV1 and CGRP). In conclusion, TRPV1 activation plays a critical role in the generation of bone cancer pain, and bone cancer increases TRPV1 expression within distinct subpopulation of DRG neurons. These findings may lead to novel strategies for the treatment of bone cancer pain.

Down-regulation of mu opioid receptor expression within distinct subpopulations of dorsal root ganglion neurons in a murine model of bone cancer pain.

Although micro opioid receptor (MOR) agonists are used for treatment of most types of pain, a recent study has suggested that the sensitivity of bone cancer pain to systemic morphine was lower than that of inflammatory pain. However, the reasons for this have remained unclear. In this study, MOR expression and the analgesic effects of morphine in a bone cancer model were compared with those in an inflammatory pain model. A bone cancer pain model and an inflammatory pain model were made by implantation of sarcoma cells into the intramedullary space of the femur and hind-paw injection of complete Freunds adjuvant (CFA), respectively. In a behavioral study, sarcoma-implanted mice showed flinching behavior of magnitude comparable to that induced by CFA injection. The flinching behavior of sarcoma-implanted mice was less sensitive to intrathecal morphine than that of CFA-injected mice. Western blot analysis showed that MOR expression in the dorsal root ganglion (DRG) ipsilateral to sarcoma implantation was significantly reduced, while that in the DRG ipsilateral to CFA injection was increased. In sarcoma-implanted mice, the percentage of MOR-positive DRG neuronal profiles was lower than that in control mice (30.3% vs. 45.2%). In particular, MOR expression was reduced among calcitonin gene-related peptide- and transient receptor potential vanilloid subfamily 1-positive DRG neuronal profiles, which are considered to be involved in the generation of bone cancer pain (from 61.5% to 41.5% and from 72.1% to 48.4%, respectively). These results suggest that down-regulation of MOR in the distinct populations of DRG neurons contributes to the fact that higher doses of morphine are needed to produce analgesia in bone cancer as compared with those used in non-malignant inflammatory situations.

FORMALIN-INDUCED NOCICEPTION ACTIVATES A MONOAMINERGIC DESCENDING INHIBITORY SYSTEM

Neural plasticity of afferent pain pathways that is induced by prolonged or repeated noxious stimuli may contribute to activate intrinsic inhibitory mechanisms in CNS. In order to clarify the role of the monoaminergic descending inhibitory system in acute nociception and inflammatory pain, we examined if this inhibitory system would modulate the tonic response to formalin-induced nociception. Yohimbine, alpha2 adrenergic antagonist, or methysergide, serotonin antagonist was administered intrathecally before or after subcutaneous 2% formalin injection into the plantar of the hind paw in rats. In another series of the experiment, the tissue of the spinal dorsal half of the untreated rats and post-formalin-treated rats were sampled and analyses of monoamine levels were carried out by HPLC. The subcutaneous formalin evoked biphasic flinching behavior of the injected paw. Intrathecal pretreatment with yohimbine and methysergide produced a significantly greater increase in the number of flinches than in the control in phase 1, intermediate period and phase 2. Posttreatment with yohimbine and methysergide showed a significantly greater increase in the number of flinches in phase 2. Furthermore, formalin injection induced significant increases in noradrenaline, MHPG, serotonin (5-hydroxytryptamine; 5-HT) and 5-HIAA concentrations in both the ipsi- and contralateral dorsal halves. These results suggest that the pain state produced by formalin-induced chemical and/or inflammatory nociception is under the modulation of the monoaminergic (noradrenergic and serotonergic) descending inhibitory system.

Premedication with oral dextromethorphan reduces postoperative pain after tonsillectomy

The aim of the present study was to examine whether premedication with dextromethorphan, a clinically available N-methyl-D-aspartic acid (NMDA) receptor antagonist, could reduce postoperative pain after tonsillectomy. Thirty-six patients scheduled for elective bilateral tonsillectomy were investigated in a double-blinded, randomized study. The patients were randomly assigned to one of three groups: control, dextromethorphan 30 mg (Dex 30), and dextromethorphan 45 mg (Dex 45) groups. In the control group, premedication was with oral placebo and intramuscular (IM) midazolam and atropine. In the Dex 30 and Dex 45 groups, patients were premedicated with IM midazolam and atropine and oral dextromethorphan 30 mg and 45 mg, respectively. Pain was evaluated repeatedly throughout 7 postoperative days, at rest and on swallowing, using a self-rating visual analog scale (VAS). The total doses of analgesics administered postoperatively were also recorded. The Dex 45 group showed significantly lower VAS scores than the control group both at rest and on swallowing throughout the 7 days. The total doses of postoperative analgesics in the Dex 45 group were significantly less than those in the control group. The Dex 30 group showed significantly lower VAS scores than the control group at rest, but not on swallowing. These results indicate that premedication with Dex 45 reduces postoperative pain after tonsillectomy, not only at rest but on swallowing. Implications: Recently, it has been suggested that central sensitization caused by the activation of N-methyl-D-aspartic acid receptors may contribute to the postoperative pain. We found that premedication with 45 mg of dextromethorphan, a clinically available N-methyl-D-aspartic acid receptor antagonist, reduced postoperative pain after tonsillectomy. (Anesth Analg 1998;86:594-7)

Analgesic mechanisms of ketamine in the presence and absence of peripheral inflammation

Background The studies on the mechanisms of ketamine antinociception have led to conflicting results. In this study, the authors investigated the contribution of supraspinal monoaminergic descending inhibitory system to ketamine analgesia for acute nociception and inflammation-induced hyperalgesia. Methods Male Sprague-Dawley rats were used. The paw withdrawal latencies to radiant heat stimuli were measured to assess the thermal nociceptive threshold. The analgesic effects of intrathecal or intraperitoneal ketamine were examined in the rats that received unilateral intraplantar carrageenan and in those that were untreated. In addition, it was examined whether pretreatment with intrathecal yohimbine or methysergide inhibited the analgesic effects of ketamine. Using an intrathecal microdialysis method, noradrenaline and 5-hydroxytryptamine concentrations in lumbar cerebrospinal fluid were measured after intraperitoneal ketamine in both saline- and carrageenan-treated rats. Results In the untreated rats, intraperitoneal but not intrathecal ketamine produced antinociceptive effects in a dose-dependent manner. Pretreatment with intrathecal yohimbine or methysergide inhibited these antinociceptive effects. Intraplantar carrageenan significantly reduced paw withdrawal latencies on the injected paw but not on the contralateral paw. Both intraperitoneal and intrathecal ketamine reversed the shortened paw withdrawal latencies on the injected side in a dose-dependent manner without any effects on the contralateral side. Neither yohimbine nor methysergide inhibited these antihyperalgesic effects. In analyses of monoamines, the magnitude of increase in monoamines after intraperitoneal ketamine was significantly smaller in the carrageenan-treated rats than in the saline-treated rats. Conclusion These results demonstrated that ketamine produced antinociceptive effects through an activation of the monoaminergic descending inhibitory system, whereas, in a unilateral peripheral inflammation-induced hyperalgesic state, the monoaminergic system did not contribute to the antihyperalgesic effects of ketamine. The mechanisms of the antinociceptive and antihyperalgesic properties of ketamine are different.

SB366791, a TRPV1 antagonist, potentiates analgesic effects of systemic morphine in a murine model of bone cancer pain

BACKGROUND Bone cancer pain has a major impact on the quality of life of cancer patients but is difficult to treat. Therefore, development of a novel strategy for bone cancer pain is needed for improvement of the patient quality of life. In this study, we examined the analgesic effects of the combination of a transient receptor potential vanilloid subfamily 1 (TRPV1) antagonist and morphine on pain-related behaviours in a murine model of bone cancer pain. METHODS C3H/HeJ mice underwent injection of osteolytic sarcoma cells into the intramedullary space of the femur. The analgesic effects of intraperitoneal morphine and the analgesic effect of a TRPV1 antagonist, SB366791 [N-(3-methoxyphenyl)-4-chlorocinnamide], on bone cancer pain-related behaviours were examined. The analgesic effects of the combination of SB366791 and morphine on bone cancer pain were also examined. RESULTS Intraperitoneal morphine significantly reduced the number of spontaneous flinches and improved ambulation only at the highest dose of 10 mg kg(-1) whereas weight-bearing was not improved. Intraperitoneal SB366791 at doses of 0.3 and 1.0 mg kg(-1), but not at a dose of 0.1 mg kg(-1), reduced the number of spontaneous flinches, whereas neither weight-bearing nor ambulation was improved. Addition of a sub-analgesic dose of SB366791 (0.1 mg kg(-1)) to morphine significantly reduced the number of flinches and improved weight-bearing compared with the effects of morphine alone. CONCLUSIONS Our findings showed that the combination of morphine and SB366791 has potent analgesic effects on bone cancer pain. The findings of this study may lead to novel strategies for the treatment of bone cancer pain.

The effects of peripheral administration of a novel selective antagonist for prostaglandin E receptor subtype EP1, ONO-8711, in a rat model of postoperative pain

Mechanically evoked pain, also known as incident pain, induced by coughing or deep breathing after surgery leads to potentially devastating consequences. It is generally thought that the prostaglandin receptor- (especially, the receptor for prostaglandin E2, EP receptor) mediated sensitization of sensory nerve fibers is a key contributor to the generation of hyperalgesia. We examined whether a peripherally administered novel selective EP1 antagonist, ONO-8711, would be a potential analgesic for incision-induced mechanical hyperalgesia. We used a rat model of postoperative pain introduced by Brennan et al. (1). Withdrawal thresholds to punctate stimulation and response frequencies to nonpunctate mechanical stimulation were determined by using von Frey filaments applied adjacent to the wound and directly to the incision site of the hind paw, respectively. Mechanical hyperalgesia to punctate and nonpunctate stimuli was observed 2 and 24 h after the incision. ONO-8711 (2, 10, or 50 &mgr;g) or saline was administered subcutaneously into the hind paw on the ipsilateral side to the incision. ONO-8711 significantly (P < 0.01) increased the withdrawal thresholds to punctate mechanical stimulation and significantly (P < 0.01) decreased the response frequencies to nonpunctate mechanical stimulation in a dose- and time-dependent manner 2 and 24 h after the incision. We conclude that EP1 receptor-mediated sensitization of sensory nerve fibers may contribute to the generation of mechanical hyperalgesia produced by incisional surgery, and that the EP1 receptor antagonist ONO-8711 may be an option for treatment of postoperative pain, especially incident pain. Implications The peripheral administration of an antagonist for EP1 receptor that is a subtype of prostaglandin E receptors can inhibit the mechanical hyperalgesia induced by a surgical incision.