ro-25-6981 and Neuralgia

Interneurons operating with glycine neurotransmitter are involved in the regulation of pain transmission in the dorsal horn of the spinal cord. In addition to interneurons, glycine release also occurs from glial cells neighboring glutamatergic synapses in the spinal cord. Neuronal and glial release of glycine is controlled by glycine transporters (GlyTs). Inhibitors of the two isoforms of GlyTs, the astrocytic type-1 (GlyT-1) and the neuronal type-2 (GlyT-2), decrease pain sensation evoked by injuries of peripheral sensory neurons or inflammation. The function of dorsal horn glycinergic interneurons has been suggested to be reduced in neuropathic pain, which can be reversed by GlyT-2 inhibitors (Org-25543, ALX1393). Several lines of evidence also support that peripheral nerve damage or inflammation may shift glutamatergic neurochemical transmission from N-methyl-D aspartate (NMDA) NR1/NR2A receptor- to NR1/NR2B receptor-mediated events (subunit switch). This pathological overactivation of NR1/NR2B receptors can be reduced by GlyT-1 inhibitors (NFPS, Org-25935), which decrease excessive glycine release from astroglial cells or by selective antagonists of NR2B subunits (ifenprodil, Ro 25-6981). Although several experiments suggest that GlyT inhibitors may represent a novel strategy in the control of neuropathic pain, proving this concept in human beings is hampered by lack of clinically applicable GlyT inhibitors. We also suggest that drugs inhibiting both GlyT-1 and GlyT-2 non-selectively and reversibly, may favorably target neuropathic pain. In this paper we overview inhibitors of the two isoforms of GlyTs as well as the effects of these drugs in experimental models of neuropathic pain. In addition, the possible mechanisms of action of the GlyT inhibitors, i.e. how they affect the neurochemical and pain transmission in the spinal cord, are also discussed. The growing evidence for the possible therapeutic intervention of neuropathic pain by GlyT inhibitors further urges development of drugable compounds, which may beneficially restore impaired pain transmission in various neuropathic conditions.

Topics: Animals; Glycine; Glycine Plasma Membrane Transport Proteins; Humans; Hyperalgesia; Neuralgia; Neuroglia; Neurons; Phenols; Piperidines; Receptors, N-Methyl-D-Aspartate; Serine; Spinal Cord Dorsal Horn; Synapses; Synaptic Transmission

Postsynaptic density-95 (PSD-95) is a synaptic scaffolding protein that plays a crucial role in the development of neuropathic pain. However, the underlying mechanism remains unclear. To address the role of PSD-95 in N-methyl-D-aspartate receptor subtype 2B (NR2B) -mediated chronic pain, we investigated the relationship between PSD-95 activation and NR2B function in the spinal cord, by using a rat model of sciatic nerve chronic constriction injury (CCI). We demonstrate that the expression levels of total PSD-95 and cAMP response element binding protein (CREB), as well as phosphorylated NR2B, PSD-95, and CREB, in the spinal dorsal horn, and the interaction of NR2B with PSD-95 were increased in the CCI animals. Intrathecal injection of the selective NR2B antagonist Ro 25-6981 increased paw withdrawal latency, in a thermal pain assessment test. Moreover, repeated treatment with Ro 25-6981 markedly attenuated the thermal hypersensitivity, and inhibited the CCI-induced upregulation of PSD-95 in the spinal dorsal horn. Furthermore, intrathecal injection of the PSD-95 inhibitor strikingly reversed the thermal and mechanical hyperalgesia. Our results suggest that blocking of NR2B signaling in the spinal cord could be used as a therapeutic candidate for treating neuropathic pain.

Topics: Animals; Behavior, Animal; CREB-Binding Protein; Disease Models, Animal; Disks Large Homolog 4 Protein; Hyperalgesia; Injections, Spinal; Male; Neuralgia; Phenols; Phosphorylation; Piperidines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Sciatic Nerve; Signal Transduction; Spinal Cord Dorsal Horn; Up-Regulation

Neuropathic pain is associated with abnormal sensitivity of the central nervous system. Although the mechanism underlying the development of sensitization remains to be fully elucidated, recent studies have reported that neuroplastic changes in the pain circuitry may be involved in hypersensitivity associated with neuropathic pain. However, it is difficult to investigate such phenomena in existing animal pain model. Therefore, in this study, we developed a novel animal model - the circuit plasticity reconstruction (CPR) model - to mimic central sensitization associated with neuroplastic changes.. NMDA and Ro 25-6981 were injected into the right insular cortex of Sprague-Dawley rats, while electrical stimulation was delivered to the contralateral hind paw. Mechanical allodynia was tested by von Frey test with up-down method, and neuroplastic changes were confirmed by PSA-NCAM-positive immunostaining.. The mechanical withdrawal threshold of the left hind paw decreased beginning 1 day after CPR modelling and persisted until day 21 comparing to the modified CPR 1 (mod-CPR 1) group (CPR: 91.68 ± 1.8%, mod-CPR 1: 42.71 ± 3.4%, p < 0.001). In contrast, mod-CPR 2 surgery without electrical stimulation did not induce mechanical allodynia. Immunostaining for PSA-NCAM also revealed that neuroplastic changes had occurred in the CPR group.. Our results demonstrated that CPR modelling induced neuroplasticity within the insular cortex, leading to alterations in the neural circuitry and central sensitization.. This article represents that the CPR model can mimic the neuropathic pain derived by neuroplastic changes. Our findings indicate that the CPR model may aid the development of novel therapeutic strategies for neuropathic pain and in elucidating the mechanisms underlying pain induced by central sensitization and neuroplastic changes.

Topics: Animals; Central Nervous System Sensitization; Cerebral Cortex; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Agonists; Male; N-Methylaspartate; Neural Cell Adhesion Molecule L1; Neuralgia; Neuronal Plasticity; Pain Threshold; Phenols; Piperidines; Rats; Rats, Sprague-Dawley; Sialic Acids

N-Methyl-d-aspartate (NMDA) receptors are thought to play an important role in the processes of central sensitization and pathogenesis of neuropathic pain, particularly after spinal cord injury (SCI). NMDA antagonists effectively reduce neuropathic pain, but serious side effects prevent their use as therapeutic drugs. NMDA NR2B antagonists have been reported to effectively reduce inflammatory and neuropathic pain. In this study, we investigated the effects of NR2B antagonists on neuropathic pain and the expression of NR2B in the spinal cord in 2 SCI models. SCI was induced at T12 by a New York University impactor (contusion) or by sectioning of the lateral half of the spinal cord (hemisection). Ifenprodil (100, 200, 500, 1000nmol) and Ro25-6981 (20, 50, 100, 200nmol) were intrathecally injected and behavioral tests were conducted. Ifenprodil increased the paw withdrawal threshold in both models but also produced mild motor depression at higher doses. Ro25-6981 increased the mechanical nociceptive threshold in a dose-dependent manner without motor depression. NR2B expression was significantly increased on both sides at the spinal segments of L1-2 and L4-5 in the hemisection model but did not change in the contusion model. Increased expression of NR2B in the hemisection model was reduced by intrathecal ifenprodil. These results suggest that intrathecal NMDA NR2B antagonist increased the mechanical nociceptive threshold after SCI without motor depression. A selective subtype of NMDA receptor, such as NR2B, may be a more selective target for pain control because NMDA receptors play a crucial role in the development and maintenance of chronic pain.

Topics: Animals; Behavior, Animal; Excitatory Amino Acid Antagonists; Male; Neuralgia; Pain Measurement; Phenols; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Spinal Cord Injuries

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

Topics: Animals; Astrocytes; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Guanylate Cyclase; Hyperalgesia; Indazoles; JNK Mitogen-Activated Protein Kinases; Male; Neuralgia; Neurons; Nitric Oxide Synthase Type I; Pain Measurement; Phenols; Phosphorylation; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Spinal Cord

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24-48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0-48 h) (r=-0.974, p=0.001), but not late stage (3-28 days) (r=0.3395, p=0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24-48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24-48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3-14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0-48 h), while the activation of NMDA-2B receptors

Topics: Analysis of Variance; Animals; Brain-Derived Neurotrophic Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hyperalgesia; Male; Neuralgia; Pain Measurement; Phenols; Piperidines; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptor, trkB; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Spinal Cord Injuries; Statistics as Topic; Time Factors

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn has been shown to be essential for the initiation of central sensitization and the hyperexcitability of dorsal horn neurons in chronic pain. However, whether the spinal NR2B-containing NMDA (NMDA-2B) receptors are involved still remains largely unclear. Using behavioral test and in vivo extracellular electrophysiological recording in L5 spinal nerve-ligated (SNL) neuropathic rats, we investigate the roles of spinal cord NMDA-2B receptors in the development of neuropathic pain. Our study showed that intrathecal (i.t.) injection of Ro 25-6981, a selective NMDA-2B receptor antagonist, had a dose-dependent anti-allodynic effect without causing motor dysfunction. Furthermore, i.t. application of another NMDA-2B receptor antagonist ifenprodil prior to SNL also significantly inhibited the mechanical allodynia but not the thermal hyperalgesia. These data suggest that NMDA-2B receptors at the spinal cord level play an important role in the development of neuropathic pain, especially at the early stage following nerve injury. In addition, spinal administration of Ro 25-6981 not only had a dose-dependent inhibitory effect on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in both normal and SNL rats, but also significantly inhibited the long-term potentiation (LTP) in the C-fiber responses of WDR neurons induced by high-frequency stimulation (HFS) applied to the sciatic nerve. These results indicate that activation of the dorsal horn NMDA-2B receptors may be crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability following nerve injury. In conclusion, the spinal cord NMDA-2B receptors play a role in the development of central sensitization and neuropathic pain via the induction of LTP in dorsal horn nociceptive synaptic transmission. Therefore, the spinal cord NMDA-2B receptor is likely to be a target for clinical pain therapy.

Topics: Action Potentials; Analysis of Variance; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Hyperalgesia; Long-Term Potentiation; Male; Motor Activity; Nerve Fibers; Neuralgia; Pain Measurement; Pain Threshold; Phenols; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sensory Receptor Cells; Spinal Cord; Time Factors