Masaki Toriyabe

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.

Contribution of interaction between nitric oxide and cyclooxygenases to the production of prostaglandins in carrageenan-induced inflammation

Background:Nitric oxide (NO) and prostaglandins (PGs) are crucial mediators contributing to generation of inflammatory responses and pain. This study was designed to investigate the effects of peripherally released NO on cyclooxygenase (COX) expression/activation and production of PGs in carrageenan-induced inflammation. Methods:A microdialysis probe was implanted subcutaneously into the skin of hind paws of rats. The concentrations of NO metabolites, PGE2, and 6-keto-PGF1&agr; (metabolite of PGI2) in the dialysate were measured. Carrageenan was injected into the plantar surface of the hind paw during perfusion of the dialysis catheter with modified Ringer’s solution or NG-monomethyl-l-arginine acetate. In addition, the effects of the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitor NS-398 on the production of NO, PGE2, and 6-keto-PGF1&agr; were examined. Western blotting was performed to evaluate the expression of COX-1 and COX-2 in the skin at the site of the inflammation. Results:Carrageenan injection resulted in increases in the concentrations of NO, PGE2, and PGI2, and these increases were completely suppressed by NG-monomethyl-l-arginine acetate. SC-560 effectively inhibited the increase in PGE2 and PGI2 concentrations for the first 2 h, and NS-398 inhibited 3–6 h after carrageenan. Western blot analysis showed that the concentrations of both COX-1 and COX-2 in the skin increased after carrageenan. The up-regulation of COX-1 in the skin was observed 3 and 6 h after carrageenan and was not suppressed in the rats treated with NG-monomethyl-l-arginine acetate. The up-regulation of COX-2 in the skin was also observed 3 and 6 h after carrageenan and was completely suppressed in the rats treated with NG-monomethyl-l-arginine acetate. Conclusion:The results of the current study suggest that NO activates COX-1 in the early phase of carrageenan and up-regulates COX-2 expression in the late phase in the skin, resulting in production of PGE2 and PGI2 at the site of inflammation, which would contribute to exacerbation of the inflammatory process.

The activation of 5-HT3 receptors evokes GABA release in the spinal cord

We carried out experiments using a microdialysis method to determine whether activation of 5-HT(3) receptors increases the concentration of GABA in the dorsal horn of the spinal cord. Intrathecal perfusion of a selective 5-HT(3) receptor agonist, 1-phenylbiguanide (0.3, 1.0, and 3.0 mM) dose-dependently, increased GABA concentration, while concentrations of glutamate and glycine were not changed. The concentration of aspartate was increased only by 3.0 mM of 1-phenylbiguanide. Our results have provided direct evidence that activation of 5-HT(3) receptors evokes GABA release in the spinal dorsal horn, possibly producing analgesia.

Intracerebroventricular morphine produces antinociception by evoking γ-aminobutyric acid release through activation of 5-hydroxytryptamine 3 receptors in the spinal cord

Background It has been generally considered that supraspinal morphine activates the serotonergic descending inhibitory system and releases serotonin (5-hydroxytryptamine [5-HT]) in the spinal cord, producing antinociception through activation of 5-HT receptors. The involvement of a spinal &ggr;-aminobutyric acid–mediated (GABAergic) system is also suggested in supraspinal morphine antinociception. It has been reported that spinal GABAergic system contributes to 5-HT3 receptor-mediated antinociception. In this study, the authors investigated the contribution of spinal 5-HT3 receptor and the GABAergic system in the intracerebroventricular morphine–induced antinociception. Methods Male Sprague-Dawley rats were used. Using the spinal microdialysis method, concentrations of 5-HT and GABA were measured after intracerebroventricular morphine administration. The effect of intracerebroventricular naloxone or spinal perfusion of a selective 5-HT3 receptor antagonist 3-tropanyl-indole-3-carboxylate methiodide on the spinal release of GABA after intracerebroventricular morphine administration was also examined. In the behavioral study, involvement of 5-HT3 receptors or GABAA receptors in the intracerebroventricular morphine–induced antinociceptive effect was investigated using the tail-flick test. Results Intracerebroventricular morphine (40 nmol) significantly increased spinal GABA and 5-HT release. Evoked spinal GABA release was reversed by intracerebroventricular naloxone (40 nmol) or spinal perfusion of 3-tropanyl-indole-3-carboxylate methiodide (1 mm). In the behavioral study, intracerebroventricular morphine produced significant antinociception. Intrathecal administration of either GABAA receptor antagonist bicuculine or 3-tropanyl-indole-3-carboxylate methiodide but not vehicle reversed the morphine-induced antinociceptive effect. Conclusion Intracerebroventricular morphine evokes spinal GABA release via the activation of 5-HT3 receptors in the spinal cord, resulting in antinociceptive effect.

Antihyperalgesic and side effects of intrathecal clonidine and tizanidine in a rat model of neuropathic pain.

Background Although intrathecal clonidine produces pronounced analgesia, antinociceptive doses of intrathecal clonidine produce several side effects, including hypotension, bradycardia, and sedation. Intrathecal tizanidine, another &agr;2-adrenergic agonist, has provided antinociception without producing pronounced hemodynamic changes in animal studies. However, it has been unclear whether antihyperalgesic doses of intrathecal clonidine and tizanidine produce hypotension and bradycardia in a neuropathic pain state. This study was designed to evaluate the antihyperalgesic effects and side effects of intrathecal clonidine and tizanidine in a rat model of neuropathic pain. Methods Male Sprague-Dawley rats were chronically implanted with lumbar intrathecal catheters, and the sciatic nerve was loosely ligated. After 21–28 days after surgery, the rats received intrathecal clonidine (0.3, 1.0, and 3.0 &mgr;g) and tizanidine (1.0, 2.0, and 5.0 &mgr;g), and the antihyperalgesic effects of thermal and mechanical stimuli were examined. In addition, the changes in blood pressure and heart rate, sedation level, and other side effects after intrathecal administration of drugs were recorded. Results The administration of 3.0 &mgr;g intrathecal clonidine or 5.0 &mgr;g tizanidine significantly reversed both thermal and mechanical hyperalgesia. The administration of 3.0 &mgr;g intrathecal clonidine, but not 5.0 &mgr;g tizanidine, significantly decreased mean blood pressure and heart rate and produced urinary voiding. A greater sedative effect was produced by 3.0 &mgr;g intrathecal clonidine than by 5.0 &mgr;g tizanidine. Conclusion The antihyperalgesic dose of intrathecal clonidine and the antinociceptive doses produced several side effects. Intrathecal tizanidine at the dose that reversed hyperalgesia would be preferable for neuropathic pain management because of absence of hypotension and bradycardia and lower incidence of sedation.

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.

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.

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.