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 Freund's 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.

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.

Immunohistochemical analysis of acid-sensing ion channel 2 expression in rat dorsal root ganglion and effects of axotomy.

Several studies have suggested that acid-sensing ion channel 2 (ASIC2) plays a role in mechanoperception and acid sensing in the peripheral nervous system. We examined the expression and distribution of ASIC2 in the rat dorsal root ganglion, the co-localization of ASIC2 with tropomyosin-related kinase (trk) receptors, and the effects of axotomy on ASIC2 expression. ASIC2 immunoreactivity was observed in both neurons and satellite cells. ASIC2-positive neurons accounted for 16.5 +/- 2.4% of the total neurons in normal dorsal root ganglion. Most ASIC2-positive neurons were medium-to-large neurons and were labeled with neurofilament 200 kD (NF200). Within these neurons, ASIC2 was not evenly distributed throughout the cytoplasm, but rather was accumulated prominently in the cytoplasm adjacent to the axon hillock and axonal process. We next examined the co-localization of ASIC2 with trk receptors. trkA was expressed in few ASIC2-positive neurons, and trkB and trkC were observed in 85.2% and 53.4% of ASIC2-positive neurons, respectively, while only 6.9% of ASIC2-positive neurons were co-localized with trkC alone. Peripheral axotomy markedly reduced ASIC2 expression in the axotomized dorsal root ganglion neurons. On the other hand, intense ASIC2 staining was observed in satellite cells. These results show that ASIC2 is expressed in the distinct neurochemical population of sensory neurons as well as satellite cells, and that peripheral axotomy induced marked reductions in ASIC2 in neurons.

Effects of thiopental on bispectral index and heart rate variability.

BACKGROUND AND OBJECTIVE: Thiopental has been reported to reduce sympathetic tone, however, it is not clear whether change in heart rate variability is associated with depth of anaesthesia. The purpose of the present study was to evaluate changes in heart rate variability at different depths of hypnosis during induction of anaesthesia with thiopental. METHODS: We studied 17 ASA I patients scheduled for minor surgery. The depth of hypnosis was monitored by the BIS. Spectral analysis of heart rate variability using a maximum entropy method resulted in a characteristic power spectrum with two main regions, a high frequency and a low frequency. Haemodynamics, entropy, low frequency, high frequency and low frequency/high frequency were monitored in an awake state and after the induction of anaesthesia. RESULTS: Heart rate increased in a BIS-dependent manner, whereas blood pressure showed no significant changes during the study period. High frequency, entropy and low frequency decreased with a reduction in the BIS value. Low frequency/high frequency showed no significant change during the study period. CONCLUSIONS: Induction of anaesthesia with thiopental increased heart rate and decreased high frequency, entropy and low frequency in a BIS-dependent manner, indicating that thiopental reduces cardiac parasympathetic tone depending on the depth of hypnosis.

Endothelin-1 enhances capsaicin-evoked intracellular Ca2+ response via activation of endothelin a receptor in a protein kinase Cepsilon-dependent manner in dorsal root ganglion neurons.

Increasing evidence indicates that endothelin-1 has a role for peripheral nociceptive signaling in animals and humans. However, the mechanisms of the nociceptive effects of endothelin-1 have not been fully understood. The current study investigated the effects of endothelin-1 on the capsaicin-evoked intracellular Ca2+ response of cultured adult mice dorsal root ganglion neurons. Dorsal root ganglia were harvested from adult male C57B6N mice and were cultured. With a digital image analysis system, we detected the [Ca2+]i image of cultured dorsal root ganglion cells after loading with Fura-2 acetoxymethyl. In addition, co-localization of protein kinase Cepsilon with transient receptor potential V1 and the translocation of protein kinase Cepsilon were investigated using immunohistochemical methods. Endothelin-1 (10 nM) enhanced an increase in [Ca2+]i by capsaicin (10 nM) from 87.6+/-11.6 nM to 414.8+/-62.3 nM (71 of 156 neurons). The inhibition of endothelin A receptor (BQ-123) significantly suppressed the enhancing effect of endothelin-1. In addition, a nonselective protein kinase C inhibitor (bisindolylmaleimide I) significantly suppressed the enhancing effect of endothelin-1. A myristoyl-tagged membrane-permeant-protein kinase Cepsilon V1-2 inhibitory peptide also significantly suppressed the enhancing effect of endothelin-1. In the immunocytochemical study, protein kinase Cepsilon immunoreactivity was found in most of transient receptor potential V1-positive neurons. After endothelin-1 application, protein kinase Cepsilon immunoreactivity was observed to be translocated from the cytosol to the cell membrane in transient receptor potential V1-positive neurons. Our results indicate that endothelin-1 enhances the response of dorsal root ganglion neurons to capsaicin in a protein kinase Cepsilon-dependent manner. Our findings may lead to a new strategy to treat pain associated with endothelin-1.

Recovery characteristics and post-operative delirium after long-duration laparoscope-assisted surgery in elderly patients: propofol-based vs. sevoflurane-based anesthesia.

BACKGROUND: Post-operative mental dysfunction may be an important problem in elderly patients. This study was designed to compare the effects of propofol and sevoflurane anesthesia on recovery characteristics and the incidence of post-operative delirium (POD) in long-duration laparoscopic surgery for elderly patients. METHODS: Fifty ASA physical status I-II patients over the age of 65 scheduled for laparoscopic surgery lasting 3 h or more randomly received propofol (group P, n = 25) or sevoflurane (group S, n = 25) for both induction and maintenance of general anesthesia. Both groups were combined with continuous perioperative epidural analgesia. The level of primary anesthetics was adjusted to maintain changes in mean arterial pressure within 20% of the pre-anesthetic values. The emergence times from anesthesia (eye opening, extubation, response to command, and orientation) were recorded, and the occurrence of POD was assessed by the delirium rating scale (DRS) during the first 3 days after surgery. All patients received oxygen and continuous epidural analgesia postoperatively. RESULTS: Immediate emergence, i.e. eye opening and extubation was significantly faster after sevoflurane (P < 0.05). There was no significant difference between the incidences of POD in the two groups during the first 3 days after surgery. The scores for DRS on day 2 and 3 after surgery, however, were significantly higher in group P than in group S (P < 0.01). CONCLUSION: Sevoflurane may be preferable to propofol for general anesthesia in combination with epidural analgesia with respect to less effect on mental function during the early postoperative period for long-duration laparoscopic surgery in elderly patients.

Pneumocephalus following an epidural blood patch.

Pneumocephalus is a rare complication of epidural block. We report a case of pneumocephalus complicating an epidural blood patch performed 3 days after unintentional dural puncture. Pneumocephalus may occur during an epidural blood patch procedure, even if the epidural needle tip is within the epidural space.

Involvement of capsaicin-sensitive fibers in spinal NMDA-induced glutamate release.

The activation of spinal NMDA receptors can evoke glutamate release through the production of nitric oxide (NO) within the spinal cord, resulting in pain-relating behavior. In this study, we investigated the involvement of capsaicin-sensitive primary afferents in this phenomenon using in vivo intrathecal microdialysis. Intrathecal NMDA perfusion evoked increases in the concentrations of NO metabolises and glutamate and in pain-related behavior in both neonatal capsaicin and vehicle-treated rats. Although the degrees of increase in NO metabolises in capsaicin- and vehicle-treated rats were not significantly different, capsaicin-treated rats showed significantly smaller increases in glutamate concentration and pain-related behavior than did vehicle-treated rats. Our results showed that glutamate release from capsaicin-sensitive primary afferent terminals is involved in spinal NMDA-induced pain.

Acute respiratory and metabolic acidosis induced by excessive muscle contraction during spinal evoked stimulation.

Spinal somatosensory evoked potentials (SSEPs) have been used to monitor spinal cord function during corrective scoliosis surgery. We report three cases in which direct epidural stimulation for measurement of SSEPs produced paraspinal muscle contraction, resulting in respiratory and metabolic acidosis. In two of the cases, SSEP-induced acidosis was observed even when only the first twitch of the train-of-four response was detectable after a second dose of muscle relaxant. In one of these two cases, the acidosis was abolished after a sufficient dose of vecuronium to ablate the twitch response. To prevent SSEP-induced respiratory and metabolic acidosis, we recommend that SSEPs should be measured only when profound neuromuscular blockade has been obtained.

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.

[The effect of continuous intra-articular and intra-bursal infusion of lidocaine on postoperative pain following shoulder arthroscopic surgery].

We evaluated the effects of continuous intra-articular and intra-bursal infusion of lidocaine on postoperative pain following shoulder arthroscopic surgery. Forty-one ASA I-II patients scheduled for shoulder arthroscopic surgery, were allocated into following four groups. The patients, after intra-articular arthroscopic surgery, either received intra-articular lidocaine (Group I, n = 10) or did not (Group III, n = 10). The patients after extra-articular arthroscopic surgery either received intra-bursal lidocaine (Group II, n = 11) or did not (Group IV, n = 10). Group I and Group II received 8 ml of 1% lidocaine intra-articularly and intra-bursally, respectively, at the end of surgery, followed by continuous infusion of 1% lidocaine at the rate of 2 ml.hr-1 for 24 hours. The intensities of postoperative pain were evaluated by Visual Analogue Scale (VAS), 2, 5, 8, 12, 18 and 24 hours after surgery, and by the number of patients' request for supplemental analgesic for 24 hours. The VAS scores and the number of analgesic requests were significantly lower (P < 0.05) in Group I than Group III, and in Group II than Group IV throughout the postoperative observation period. No adverse effects were observed during this study. We conclude that continuous intra-articular and intra-bursal infusion of lidocaine provides effective postoperative pain relief for shoulder arthroscopic surgery.

The effects of peripheral administration of a novel selective antagonist for prostaglandin E receptor subtype EP(1), ONO-8711, in a rat model of postoperative pain.

UNLABELLED: 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 E(2), 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 EP(1) 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 microg) 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 EP(1) receptor-mediated sensitization of sensory nerve fibers may contribute to the generation of mechanical hyperalgesia produced by incisional surgery, and that the EP(1) receptor antagonist ONO-8711 may be an option for treatment of postoperative pain, especially incident pain. IMPLICATIONS: The peripheral administration of an antagonist for EP(1) receptor that is a subtype of prostaglandin E receptors can inhibit the mechanical hyperalgesia induced by a surgical incision.

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.

Activation of peripheral NMDA-nitric oxide cascade in formalin test.

BACKGROUND: It has been suggested that peripheral glutamate and nitric oxide (NO) released by tissue-damaging stimuli play an important role in peripheral nociceptive transmission. This study was conducted to determine whether NO was released in the periphery after subcutaneous injection of formalin and whether the peripheral N-methyl-d-aspartate (NMDA)-NO cascade was activated. METHODS: During pentobarbital anesthesia, a microdialysis probe was implanted subcutaneously into the glabrous skin of both hind paws of Sprague-Dawley rats. After sample collection to obtain the basal level of NO metabolites (total amount of nitrite [NO2-] and nitrate [NO3-] [NO2--NO3-]), 5% formalin was injected into the plantar surface of the right hind paw during perfusion of the dialysis catheters with artificial cerebrospinal fluid (ACSF), the NO synthase inhibitor NG-monomethyl-L-arginine acetate, or the NMDA antagonist D,l-2-amino-5-phosphonovaleric acid through a microdialysis probe. Formalin also was injected in the animals that underwent sciatic nerve sectioning. In another series of experiments, NMDA was perfused through one probe. Samples for measurement of NO2--NO3- were collected and immediately analyzed using high-performance liquid chromatography. RESULTS: Subcutaneous formalin significantly increased the dialysate concentrations of NO2--NO3- (maximum increase 144 +/- 12% of baseline value 30 min after formalin administration; P < 0.05) on the side ipsilateral to the injection. Both NG-monomethyl-l-arginine acetate and D, l-2-amino-5-phosphonovaleric acid significantly (P < 0.05) suppressed the formalin-induced increases in NO2--NO3- concentration. In the rats with denervation of the sensory nerves, formalin did not change the NO2--NO3- concentration. In addition, NMDA perfusion significantly (P < 0.05) increased the concentrations of NO2--NO3-. CONCLUSION: The results of the current study show that subcutaneous formalin injection induces peripheral release of NO, the production of which is mediated by activation of NMDA receptors in the peripheral nervous system.

A comparison of hemodynamic changes after endotracheal intubation by using the lightwand device and the laryngoscope in normotensive and hypertensive patients.

We compared the effects of the lightwand technique on hemodynamic responses, time-to-intubation, number of attempts, and complications during tracheal intubation with those of direct-vision laryngoscopy in normotensive (LN and TN group; n = 20, respectively) and hypertensive (LH and TH group; n = 20, respectively) patients. Lightwand or laryngoscopic oral endotracheal intubation was performed after the induction of anesthesia with fentanyl and propofol and muscle relaxation with vecuronium. Systolic blood pressure, diastolic blood pressure, and heart rate were recorded, and the change from "before intubation" to "immediately after intubation" (DeltaP) in each variable was calculated. In normotensive patients, significantly larger DeltaP in systolic blood pressure was observed in the LN group than in the TN group (P < 0. 05). In hypertensive patients, there were no significant differences between the LH group and the TH group in DeltaP after intubation. The time-to-intubation and number of attempts in the lightwand groups were significantly longer and more frequent than those in the laryngoscope groups (P < 0.05). The number of patients who complained of hoarseness was larger in the lightwand groups than in the laryngoscope groups (P < 0.05). We conclude that the lightwand technique significantly attenuates hemodynamic changes after intubation in comparison with the laryngoscopic technique in normotensive patients. However, in hypertensive patients, there is no difference in hemodynamic changes between the two techniques.

[Clinical evaluation of accuracy of a new disposable pump (Syrinjector) for continuous epidural infusion].

We evaluated the accuracy of a new disposable pump (Coopdech Syrinjector), which employed the negative pressure made by injecting fluid into the pump for continuous epidural infusion. Thoracic epidural catheter was placed at the T6-T8 level prior to the lung surgery. The continuous epidural infusion of 0.25% bupivacaine at 2 or 3 ml.h-1 was started immediately after the surgery. The residual volume of bupivacaine was recorded. From our results, the injection speed of Syrinjector was satisfactory for clinical use of continuous epidural infusion of bupivacaine. However, when this pump is reused or the volume of the residual local anesthetics is less than 10 ml, the infusion accuracy will be potentially imprecise.

Altered expression of beta-catenin and c-erbB-2 in early gastric cancer.

To investigate the possible relationship between altered expression (loss of membranous staining or nuclear accumulation) of beta-catenin and invasion/metastasis in early gastric cancer (EGC), beta-catenin was detected immunohistochemically in 116 cases of EGC, including 86 differentiated and 30 undifferentiated carcinomas. In parallel, immunohistochemical expression of c-erbB-2 was analyzed in all EGC cases. Regardless of histological type, altered expression of beta-catenin was found in 47% of mucosal carcinomas and 89% of carcinomas with submucosal invasion (p<0.001). Of particular interest is that beta-catenin alteration was found in almost all EGCs with lymph node metastasis, even though no significant statistical comparison could be made. These results suggest that molecular changes resulting in abnormal beta-catenin expression participate in the process of submucosal invasion and metastasis. While loss of expression was preferentially observed in undifferentiated EGCs, nuclear accumulation was found exclusively in 24% of differentiated EGCs. c-erbB-2 was overexpressed in only 16% of differentiated EGCs but there was no correlation between this overexpression and invasion or metastasis. However, it is intriguing that 12 out of 14 cases with c-erbB-2 overexpression also showed altered beta-catenin expression, suggesting that both molecules are involved in the development of a certain set of differentiated EGCs.

Activation of spinal N-methyl-D-aspartate receptors stimulates a nitric oxide/cyclic guanosine 3,5-monophosphate/glutamate release cascade in nociceptive signaling.

BACKGROUND: Increasing evidence has suggested the possibility that the activation of N-methyl-D-aspartate (NMDA) receptors modulates spinal nociceptive transmission via a nitric oxide (NO)/cyclic guanosine 3',5'-monophosphate (cGMP) pathway. However, the existence and the role of an NO/cGMP pathway in the modulation of spinal nociceptive transmission has been unclear. The authors hypothesized that the activation of NMDA receptors stimulates an NO/cGMP pathway, and this pathway evokes glutamate release within the spinal cord, modulating spinal nociceptive transmission. METHODS: The authors have examined the effects of an NO synthase inhibitor and a soluble guanylate cyclase inhibitor on the concentrations of NO metabolites (NO2-/NO3-) and glutamate in the cerebrospinal fluid after intrathecal perfusion of NMDA, concomitantly observing pain-related behavior (scratching, biting, and vocalization) in unanesthetized, free-moving rats using an intrathecal microdialysis method. The contents of cGMP in the dorsal horn were also measured using enzyme immunoassay method. RESULTS: Intrathecal perfusion of NMDA produced pain-related behavior and increased glutamate and NO2-/NO3-concentrations in a dose-dependent manner. A competitive NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid, completely blocked the NMDA-induced responses. An NO synthase inhibitor, N(G)-monomethyl-L-arginine acetate, at a dose that completely blocked the increase in NO2-/NO3-, inhibited both the NMDA-induced pain-related behavior and the increase in glutamate concentration. In addition, a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one, also inhibited significantly NMDA-induced pain-related behavior and the increase in glutamate concentration. NMDA induced an increase in cGMP in the dorsal half of the spinal cord, which was blocked by N(G)-monomethyl-L-arginine acetate. CONCLUSIONS: The results of this study support the hypothesis that the activation of NMDA receptors modulated pain-related behavior via an NO/cGMP/glutamate release cascade within the spinal cord.