Induction of long-term potentiation and long-term depression is cell-type specific in the spinal cord.

The underlying mechanism of chronic pain is believed to be changes in excitability in spinal dorsal horn (DH) neurons that respond abnormally to peripheral input. Increased excitability in pain transmission neurons, and depression of inhibitory neurons, are widely recognized in the spinal cord of animal models of chronic pain. The possible occurrence of 2 parallel but opposing forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) was tested in 2 types of identified DH neurons using whole-cell patch-clamp recordings in mouse spinal cord slices. The test stimulus was applied to the sensory fibers to evoke excitatory postsynaptic currents in identified spinothalamic tract neurons (STTn) and GABAergic neurons (GABAn). Afferent conditioning stimulation (ACS) applied to primary afferent fibers with various stimulation parameters induced LTP in STTn but LTD in GABAn, regardless of stimulation parameters. These opposite responses were further confirmed by simultaneous dual patch-clamp recordings of STTn and GABAn from a single spinal cord slice. Both the LTP in STTn and the LTD in GABAn were blocked by an NMDA receptor antagonist, AP5, or an intracellular Ca chelator, BAPTA. Both the pattern and magnitude of intracellular Ca after ACS were almost identical between STTn and GABAn based on live-cell calcium imaging. The results suggest that the intense sensory input induces an NMDA receptor-dependent intracellular Ca increase in both STTn and GABAn, but produces opposing synaptic plasticity. This study shows that there is cell type-specific synaptic plasticity in the spinal DH.

Effect of antioxidant treatment on spinal GABA neurons in a neuropathic pain model in the mouse.

One feature of neuropathic pain is a reduced spinal gamma-aminobutyric acid (GABA)-ergic inhibitory function. However, the mechanisms behind this attenuation remain to be elucidated. This study investigated the involvement of reactive oxygen species in the spinal GABA neuron loss and reduced GABA neuron excitability in spinal nerve ligation (SNL) model of neuropathic pain in mice. The importance of spinal GABAergic inhibition in neuropathic pain was tested by examining the effects of intrathecally administered GABA receptor agonists and antagonists in SNL and naïve mice, respectively. The effects of SNL and antioxidant treatment on GABA neuron loss and functional changes were examined in transgenic GAD67-enhanced green fluorescent protein positive (EGFP+) mice. GABA receptor agonists transiently reversed mechanical hypersensitivity of the hind paw in SNL mice. On the other hand, GABA receptor antagonists made naïve mice mechanically hypersensitive. Stereological analysis showed that the numbers of enhanced green fluorescent protein positive (EGFP+) GABA neurons were significantly decreased in the lateral superficial laminae (I-II) on the ipsilateral L5 spinal cord after SNL. Repeated antioxidant treatments significantly reduced the pain behaviors and prevented the reduction in EGFP+ GABA neurons. The response rate of the tonic firing GABA neurons recorded from SNL mice increased with antioxidant treatment, whereas no change was seen in those recorded from naïve mice, which suggested that oxidative stress impaired some spinal GABA neuron activity in the neuropathic pain condition. Together the data suggest that neuropathic pain, at least partially, is attributed to oxidative stress, which induces both a GABA neuron loss and dysfunction of surviving GABA neurons.

Reactive oxygen species (ROS) modulate AMPA receptor phosphorylation and cell-surface localization in concert with pain-related behavior.

Sensitization of dorsal horn neurons (DHNs) in the spinal cord is dependent on pain-related synaptic plasticity and causes persistent pain. The DHN sensitization is mediated by a signal transduction pathway initiated by the activation of N-methyl-d-aspartate receptors (NMDA-Rs). Recent studies have shown that elevated levels of reactive oxygen species (ROS) and phosphorylation-dependent trafficking of GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) are a part of the signaling pathway for DHN sensitization. However, the relationship between ROS and AMPA-R phosphorylation and trafficking is not known. Thus, this study investigated the effects of ROS scavengers on the phosphorylation and cell-surface localization of GluA1 and GluA2. Intrathecal NMDA- and intradermal capsaicin-induced hyperalgesic mice were used for this study since both pain models share the NMDA-R activation-dependent DHN sensitization in the spinal cord. Our behavioral, biochemical, and immunohistochemical analyses demonstrated that: 1) NMDA-R activation in vivo increased the phosphorylation of AMPA-Rs at GluA1 (S818, S831, and S845) and GluA2 (S880) subunits; 2) NMDA-R activation in vivo increased cell-surface localization of GluA1 but decreased that of GluA2; and 3) reduction of ROS levels by ROS scavengers PBN (N-tert-butyl-α-phenylnitrone) or TEMPOL (4-hydroxy-2, 2, 6, 6-tetramethylpiperidin-1-oxyl) reversed these changes in AMPA-Rs, as well as pain-related behavior. Given that AMPA-R trafficking to the cell surface and synapse is regulated by NMDA-R activation-dependent phosphorylation of GluA1 and GluA2, our study suggests that the ROS-dependent changes in the phosphorylation and cell-surface localization of AMPA-Rs are necessary for DHN sensitization and thus, pain-related behavior. We further suggest that ROS reduction will ameliorate these molecular changes and pain.

Regulation of Wnt signaling by nociceptive input in animal models.

BACKGROUND: Central sensitization-associated synaptic plasticity in the spinal cord dorsal horn (SCDH) critically contributes to the development of chronic pain, but understanding of the underlying molecular pathways is still incomplete. Emerging evidence suggests that Wnt signaling plays a crucial role in regulation of synaptic plasticity. Little is known about the potential function of the Wnt signaling cascades in chronic pain development. RESULTS: Fluorescent immunostaining results indicate that ß-catenin, an essential protein in the canonical Wnt signaling pathway, is expressed in the superficial layers of the mouse SCDH with enrichment at synapses in lamina II. In addition, Wnt3a, a prototypic Wnt ligand that activates the canonical pathway, is also enriched in the superficial layers. Immunoblotting analysis indicates that both Wnt3a a ß-catenin are up-regulated in the SCDH of various mouse pain models created by hind-paw injection of capsaicin, intrathecal (i.t.) injection of HIV-gp120 protein or spinal nerve ligation (SNL). Furthermore, Wnt5a, a prototypic Wnt ligand for non-canonical pathways, and its receptor Ror2 are also up-regulated in the SCDH of these models. CONCLUSION: Our results suggest that Wnt signaling pathways are regulated by nociceptive input. The activation of Wnt signaling may regulate the expression of spinal central sensitization during the development of acute and chronic pain.

Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain.

The increase of cytosolic free Ca(2+) ([Ca(2+)](c)) due to NMDA receptor activation is a key step for spinal cord synaptic plasticity by altering cellular signal transduction pathways. We focus on this plasticity as a cause of persistent pain. To provide a mechanism for these classic findings, we report that [Ca(2+)](c) does not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca(2+) uptake during a [Ca(2+)](c) increase blocks induction of behavioral hyperalgesia and accompanying downstream cell signaling, with reduction of spinal long-term potentiation (LTP). Furthermore, reducing the accompanying mitochondrial superoxide levels lessens hyperalgesia and LTP induction. These results indicate that [Ca(2+)](c) requires downstream mitochondrial Ca(2+) uptake with consequent production of reactive oxygen species (ROS) for synaptic plasticity underlying chronic pain. These results suggest modifying mitochondrial Ca(2+) uptake and thus ROS as a type of chronic pain therapy that should also have broader biologic significance.

Electroacupuncture reduces the evoked responses of the spinal dorsal horn neurons in ankle-sprained rats.

Acupuncture is shown to be effective in producing analgesia in ankle sprain pain in humans and animals. To examine the underlying mechanisms of the acupuncture-induced analgesia, the effects of electroacupuncture (EA) on weight-bearing forces (WBR) of the affected foot and dorsal horn neuron activities were examined in a rat model of ankle sprain. Ankle sprain was induced manually by overextending ligaments of the left ankle in the rat. Dorsal horn neuron responses to ankle movements or compression were recorded from the lumbar spinal cord using an in vivo extracellular single unit recording setup 1 day after ankle sprain. EA was applied to the SI-6 acupoint on the right forelimb (contralateral to the sprained ankle) by trains of electrical pulses (10 Hz, 1-ms pulse width, 2-mA intensity) for 30 min. After EA, WBR of the sprained foot significantly recovered and dorsal horn neuron activities were significantly suppressed in ankle-sprained rats. However, EA produced no effect in normal rats. The inhibitory effect of EA on hyperactivities of dorsal horn neurons of ankle-sprained rats was blocked by the α-adrenoceptor antagonist phentolamine (5 mg/kg ip) but not by the opioid receptor antagonist naltrexone (10 mg/kg ip). These data suggest that EA-induced analgesia in ankle sprain pain is mediated mainly by suppressing dorsal horn neuron activities through α-adrenergic descending inhibitory systems at the spinal level.

Responses of spinal dorsal horn neurons to foot movements in rats with a sprained ankle.

Acute ankle injuries are common problems and often lead to persistent pain. To investigate the underlying mechanism of ankle sprain pain, the response properties of spinal dorsal horn neurons were examined after ankle sprain. Acute ankle sprain was induced manually by overextending the ankle of a rat hindlimb in a direction of plantarflexion and inversion. The weight-bearing ratio (WBR) of the affected foot was used as an indicator of pain. Single unit activities of dorsal horn neurons in response to plantarflexion and inversion of the foot or ankle compression were recorded from the medial part of the deep dorsal horn, laminae IV-VI, in normal and ankle-sprained rats. One day after ankle sprain, rats showed significantly reduced WBRs on the affected foot, and this reduction was partially restored by systemic morphine. The majority of deep dorsal horn neurons responded to a single ankle stimulus modality. After ankle sprain, the mean evoked response rates were significantly increased, and afterdischarges were developed in recorded dorsal horn neurons. The ankle sprain-induced enhanced evoked responses were significantly reduced by morphine, which was reversed by naltrexone. The data indicate that movement-specific dorsal horn neuron responses were enhanced after ankle sprain in a morphine-dependent manner, thus suggesting that hyperactivity of dorsal horn neurons is an underlying mechanism of pain after ankle sprain.

Reactive oxygen species contribute to neuropathic pain by reducing spinal GABA release.

Although both a loss of spinal inhibitory neurotransmission and the involvement of oxidative stress have been regarded as important mechanisms in the pathogenesis of pain, the relationship between these 2 mechanisms has not been studied. To determine whether reactive oxygen species (ROS) involvement in pain mechanisms is related to the diminished inhibitory transmission in the substantia gelatinosa (SG) of the spinal dorsal horn, behavioral studies and whole-cell recordings were performed in FVB/NJ mice. Neuropathic pain was induced by a tight ligation of the L5 spinal nerve (SNL). Pain behaviors in the affected foot were assessed by behavioral testing for mechanical hyperalgesia. Pain behaviors developed by 3 days and lasted more than 8 weeks. Both systemic and intrathecal administration of an ROS scavenger, phenyl-N-tert-butylnitrone (PBN), temporarily reversed mechanical hyperalgesia up to 2 hours, 1 week after SNL. In nonligated mice, an intrathecal injection of an ROS donor, tert-butyl hydroperoxide (t-BOOH), dose-dependently induced mechanical hyperalgesia for 1.5 hours. In whole-cell voltage clamp recordings of SG neurons, perfusion with t-BOOH significantly decreased the frequency of mIPSCs, and this effect was reversed by PBN. Furthermore, t-BOOH decreased the frequency of GABA(A) receptor-mediated mIPSCs without altering their amplitudes but did not affect glycine receptor-mediated mIPSCs. In SNL mice, mIPSC frequency in SG neurons was significantly reduced as compared with that of normal mice, which was restored by PBN. The antihyperalgesic effect of PBN on mechanical hyperalgesia was attenuated by intrathecal bicuculline, a GABA(A) receptor blocker. Our results indicate that the increased ROS in spinal cord may induce pain by reducing GABA inhibitory influence on SG neurons that are involved in pain transmission.

Involvement of reactive oxygen species in long-term potentiation in the spinal cord dorsal horn.

Recent studies suggest that reactive oxygen species (ROS) are functional messenger molecules in central sensitization, an underlying mechanism of persistent pain. Because spinal cord long-term potentiation (LTP) is the electrophysiological basis of central sensitization, this study investigates the effects of the increased or decreased spinal ROS levels on spinal cord LTP. Spinal cord LTP is induced by either brief, high-frequency stimulation (HFS) of a dorsal root at C-fiber intensity or superfusion of a ROS donor, tert-butyl hydroperoxide (t-BOOH), onto rat spinal cord slice preparations. Field excitatory postsynaptic potentials (fEPSPs) evoked by dorsal root stimulations with either Abeta- or C-fiber intensity are recorded from the superficial dorsal horn. HFS significantly increases the slope of both Abeta- and C-fiber evoked fEPSPs, thus suggesting LTP development. The induction, not the maintenance, of HFS-induced LTP is blocked by a N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5). Both the induction and maintenance of LTP of Abeta-fiber-evoked fEPSPs are inhibited by a ROS scavenger, either N-tert-butyl-alpha-phenylnitrone or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. A ROS donor, t-BOOH-induced LTP is inhibited by N-tert-butyl-alpha-phenylnitrone but not by D-AP5. Furthermore, HFS-induced LTP and t-BOOH-induced LTP occlude each other. The data suggest that elevated ROS is a downstream event of NMDA receptor activation and an essential step for potentiation of synaptic excitability in the spinal dorsal horn.

Electroacupuncture analgesia in rat ankle sprain pain model: neural mechanisms.

OBJECTIVES: Acupuncture, an alternative medical therapy with a long history, is appealing because it can activate endogenous analgesic mechanisms by minimally invasive means. The mechanisms of acupuncture, however, are not well understood yet. The following sentence was removed from our original manuscript. One of the major problems impeding understanding of the acupuncture mechanism is lack of experimental models that mimic various forms of persistent pain that respond to acupuncture in humans. METHODS: In this review, we summarize and discuss previous and recent findings regarding electroacupuncture-induced analgesia in an ankle sprain pain model and the potential underlying mechanisms of acupuncture. RESULTS: A novel model of ankle sprain pain is introduced recently and the mechanism of electroacupuncture-induced analgesia in this model has been explored. The following sentence was removed from our original manuscript. This model provides a reproducible and quantifiable index of persistent pain at the ankle joint in rats. Acupuncture at a remote site produces long-lasting and powerful analgesia. The consistent analgesic effect of acupuncture in this model has allowed us to pursue the underlying neural mechanisms. CONCLUSIONS: These studies provide insight into the mechanisms of acupuncture analgesia in one particular form of persistent pain, and hopefully will allow us to expand our knowledge to other painful conditions.

Superoxide signaling in pain is independent of nitric oxide signaling.

Two reactive oxygen species (ROS), nitric oxide (NO(.)) and superoxide ((.)O2), contribute to persistent pain. Using three different animal models where ROS mediate pain, this study examined whether NO(.) and (.)O2 converge to peroxynitrite (ONOO(-)) or whether each has an independent signaling pathway to produce hyperalgesia. The hyperalgesia after spinal nerve ligation was attenuated by removing (.)O2 by TEMPOL or inhibiting NO(.) production by L-NAME, but not by removing peroxynitrite with FeTMPyP. Nitric oxide-induced hyperalgesia was not affected by removing (.)O2 but was reduced by a guanyl cyclase inhibitor. Superoxide-induced hyperalgesia was not affected by inhibiting NO(.) production but was suppressed by a protein kinase C inhibitor. The data suggest that NO(.) and (.)O2 operate independently to generate pain.

Electroacupuncture suppresses capsaicin-induced secondary hyperalgesia through an endogenous spinal opioid mechanism.

Central sensitization, caused either by tissue inflammation or peripheral nerve injury, plays an important role in persistent pain. An animal model of capsaicin-induced pain has well-defined peripheral and central sensitization components, thus is useful for studying the analgesic effect on two separate components. The focus of this study is to examine the analgesic effects of electroacupuncture (EA) on capsaicin-induced secondary hyperalgesia, which represents central sensitization. Capsaicin (0.1%, 20 microl) was injected into the plantar side of the left hind paw, and foot withdrawal thresholds in response to von Frey stimuli (mechanical sensitivity) were determined for both primary and secondary hyperalgesia in rats. EA (2 Hz, 3 mA) was applied to various pairs of acupoints, GB30-GB34, BL40-BL60, GV2-GV6, LI3-LI6 and SI3-TE8, for 30 min under isoflurane anesthesia and then the effect of EA on mechanical sensitivity of paw was determined. EA applied to the ipsilateral SI3-TE8, but to none of the other acupoints, significantly reduced capsaicin-induced secondary hyperalgesia but not primary hyperalgesia. EA analgesic effect was inhibited by a systemic non-specific opioid receptor (OR) antagonist or an intrathecal mu- or delta-OR antagonist. EA analgesic effect was not affected by an intrathecal kappa-OR antagonist or systemic adrenergic receptor antagonist. This study demonstrates that EA produces a stimulation point-specific analgesic effect on capsaicin-induced secondary hyperalgesia (central sensitization), mediated by activating endogenous spinal mu- and delta-opioid receptors.

Persistent pain is dependent on spinal mitochondrial antioxidant levels.

Reactive oxygen species (ROS) scavengers have been shown to relieve persistent pain; however, the mechanism is not clearly understood. Superoxide produced from mitochondrial oxidative phosphorylation is considered the major source of ROS in neurons during excitation where mitochondrial superoxide levels are normally controlled by superoxide dismutase (SOD-2). The present study hypothesizes that capsaicin-induced secondary hyperalgesia is a consequence of superoxide build-up in spinal dorsal horn neurons and SOD-2 is a major determinant. To test this hypothesis, the spinal levels of SOD-2 activity, inactivated SOD-2 proteins, and mitochondrial superoxide were measured and correlated to the levels of capsaicin-induced secondary hyperalgesia in mice with and without SOD-2 manipulations. The data suggest that superoxide accumulation is a culprit in the abnormal sensory processing in the spinal cord in capsaicin-induced secondary hyperalgesia. Our studies also support the notion that SOD-2 nitration is a critical mechanism that maintains elevated superoxide levels in the spinal cord after capsaicin treatment. Finally, our findings suggest a therapeutic potential for the manipulation of spinal SOD-2 activity in pain conditions.

Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: the effect of mitochondrial electron transport complex inhibitors.

Scavengers of reactive oxygen species (ROS) have been shown to produce a strong antinociceptive effect on persistent pain, and mitochondria are suggested to be the main source of ROS in the spinal dorsal horn. To explore whether excessive generation of mitochondrial superoxide alone can induce pain, the effect of mitochondrial electron transport complex inhibitors on the development of mechanical hyperalgesia was examined in mice. Intrathecal injection of an electron transport complex inhibitor, antimycin A or rotenone, in normal mice resulted in a slowly developing but long-lasting and dose-dependent mechanical hyperalgesia. The levels of mechanical hyperalgesia after antimycin A, a complex III inhibitor, were higher than that with rotenone, a complex I inhibitor. A large increase of mitochondrial superoxide in the spinal dorsal horn and a strong antinociceptive effect of ROS scavengers, phenyl-N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were observed in antimycin A-treated mice. The study indicates that the enhanced production of spinal mitochondrial superoxide alone without nerve injury can produce mechanical hyperalgesia.

A surgical ankle sprain pain model in the rat: effects of morphine and indomethacin.

Ankle sprain is a frequent injury in humans that results in pain, swelling and difficulty in walking on the affected side. Currently a suitable animal model resembling human ankle sprain is lacking. Here, we describe an animal ankle sprain model induced by ankle ligament injury (ALI) in rats. Cutting combinations of the lateral ankle ligament complex produced pain, edema and difficulty of weight bearing, thereby mimicking severe (grade III) ankle sprain in humans. Analgesic compounds, morphine and indomethacin, significantly reversed the reduced weight bearing, thus indicating that reduction of weight bearing is partially due to pain. The ALI model is a new ankle sprain model that may be useful for the study of ankle sprain pain mechanisms and treatments, as well as for the screening of new analgesic drugs.

Phenyl N-t-butylnitrone, a reactive oxygen species scavenger, reduces zymosan-induced visceral pain in rats.

To examine a possible involvement of reactive oxygen species (ROS) in visceral pain, the levels of ROS in the colon and the effect of a ROS scavenger phenyl N-t-butylnitrone (PBN) on pain were examined in zymosan-induced colitis rats. Zymosan was instilled into the colon of adult rats. The electromyograms (EMGs) of abdominal muscle contractions in response to colorectal distension (CRD) were recorded as an indicator of visceral pain. After zymosan treatment, the rats showed enhanced EMG and elevated levels of H2O2 in the colon. PBN treatment (intraperitoneal, intrathecal or intracolonic) significantly reduced the enhanced EMGs induced by zymosan. The results suggest that elevated ROS in the spinal cord and the colon are involved in visceral pain.

Oxidative stress in the spinal cord is an important contributor in capsaicin-induced mechanical secondary hyperalgesia in mice.

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, thus suggesting ROS involvement in central sensitization. To investigate ROS involvement in central sensitization, the effects of ROS scavengers and donors on pain behaviors were examined in mice. Capsaicin- induced hyperalgesia was used as a pain model since it has 2 distinctive pain components, primary and secondary hyperalgesia representing peripheral and central sensitization, respectively. Capsaicin (25 microg/5 microl) was injected intradermally into the left hind foot. Foot withdrawal frequencies in response to von Frey filament stimuli were measured and used as an indicator of mechanical hyperalgesia. The production of ROS was examined by using a ROS sensitive dye, MitoSox. Mice developed primary and secondary mechanical hyperalgesia after capsaicin injection. A systemic or intrathecal post-treatment with either phenyl-N-tert-butylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL), ROS scavengers, significantly reduced secondary hyperalgesia, but not primary hyperalgesia, in a dose-dependent manner. Pretreatment with ROS scavengers also significantly reduced the magnitude and duration of capsaicin-induced secondary hyperalgesia. On the other hand, intrathecal injection of tert-butylhydroperoxide (t-BOOH, 5 microl), a ROS donor, produced a transient hyperalgesia in a dose-dependent manner. The number of MitoSox positive dorsal horn neurons was increased significantly after capsaicin treatment. This study suggests that ROS mediates the development and maintenance of capsaicin-induced hyperalgesia in mice, mainly through central sensitization and that the elevation of spinal ROS is most likely due to increased production of mitochondrial superoxides in the dorsal horn neurons.

Electroacupuncture-induced analgesia in a rat model of ankle sprain pain is mediated by spinal alpha-adrenoceptors.

In a previous study, we showed that electroacupuncture (EA) applied to the SI-6 point on the contralateral forelimb produces long-lasting and powerful analgesia in pain caused by ankle sprain in a rat model. To investigate the underlying mechanism of EA analgesia, the present study tested the effects of various antagonists on known endogenous analgesic systems in this model. Ankle sprain was induced in anesthetized rats by overextending their right ankle with repeated forceful plantar flexion and inversion of the foot. When rats developed pain behaviors (a reduction in weight-bearing of the affected hind limb), EA was applied to the SI-6 point on the contralateral forelimb for 30 min under halothane anesthesia. EA significantly improved the weight-bearing capacity of the affected hind limb for 2h, suggesting an analgesic effect. The alpha-adrenoceptor antagonist phentolamine (2mg/kg, i.p. or 30 microg, i.t.) completely blocked the EA-induced analgesia, whereas naloxone (1mg/kg, i.p.) failed to block the effect. These results suggest that EA-induced analgesia is mediated by alpha-adrenoceptor mechanisms. Further experiments showed that intrathecal administration of yohimbine, an alpha(2)-adrenergic antagonist, reduced the EA-induced analgesia in a dose-dependent manner, whereas terazosin, an alpha(1)-adrenergic antagonist, did not produce any effect. These data suggest that the analgesic effect of EA in ankle sprain pain is, at least in part, mediated by spinal alpha(2)-adrenoceptor mechanisms.

The role of reactive oxygen species in capsaicin-induced mechanical hyperalgesia and in the activities of dorsal horn neurons.

Previous findings that reactive oxygen species (ROS) are involved in neuropathic pain, mainly through spinal mechanisms, suggest that ROS may be involved in central sensitization. To investigate the possible role of ROS in central sensitization, we examined in rats the effects of ROS scavengers on capsaicin-induced secondary hyperalgesia, which is known to be mediated by central sensitization. We used two different ROS scavengers: phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL). Intradermal capsaicin injection (20 microg in 20 microl olive oil) into the hind paw produced primary and secondary hyperalgesia. A systemic administration of PBN (100mg/kg, i.p.) or TEMPOL (200mg/kg, i.p.) alleviated capsaicin-induced secondary, but not primary, hyperalgesia. Intrathecal injection of PBN (1mg inof veterinary Surgery/anesthesiology, College of veterinary Medic 50 microl saline) greatly reduced hyperalgesia, whereas intracerebroventricular or intradermal injection of PBN produced only a minor analgesic effect, suggesting that PBN takes effect mainly through the spinal cord. Electrophysiological recordings from wide dynamic range (WDR) neurons in the dorsal horn showed that intradermal capsaicin enhanced the evoked responses to peripheral stimuli; systemic PBN or TEMPOL restored the responses to normal levels. Removal of ROS thus restored the responsiveness of spinal WDR neurons to normal levels, suggesting that ROS is involved in central sensitization, at least in part by sensitizing WDR neurons.

Reactive oxygen species (ROS) are involved in enhancement of NMDA-receptor phosphorylation in animal models of pain.

Recent studies indicate that reactive oxygen species (ROS) play an important role in neuropathic pain, predominantly through spinal mechanisms. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N-methyl-d-aspartate (NMDA) receptors in the dorsal horn before and after removal of ROS with a ROS scavenger, phenyl-N-t-butyl nitrone (PBN), in animal models of pain. Tight ligation of the L5 spinal nerve was used for the neuropathic pain model and intradermal injection of capsaicin was used for the inflammatory pain model. Foot withdrawal thresholds to von Frey stimuli to the paw were measured as pain indicators. The number of neurons showing immunoreactivity to phosphorylated NMDA-receptor subunit 1 (pNR1) and the total amount of pNR1 proteins in the spinal cord were determined using immunohistochemical and Western blotting techniques, respectively. Hyperalgesia and increased pNR1 expression were observed in both neuropathic and capsaicin-treated rats. A systemic injection of PBN (100 mg/kg, i.p.) dramatically reduced hyperalgesia and blocked the enhancement of spinal pNR1 in both pain models within 1h after PBN treatment. The data suggest that ROS are involved in NMDA-receptor activation, an essential step in central sensitization, and thus contribute to neuropathic and capsaicin-induced pain.