Nerve Injury-induced Neuropathic Pain Research Articles

Lysophosphatidic acid 3 (LPA3) receptor-mediated LPA production via microglial activation underlies the initial mechanisms of nerve injury-induced neuropathic pain

Lysophosphatidic acid 3 (LPA3) receptor-mediated LPA production via microglial activation underlies the initial mechanisms of nerve injury-induced neuropathic pain

Therapeutic Targets for the Management of Peripheral Nerve Injury- Induced Neuropathic Pain

Neuropathic pain is a debilitating form of treatment resistant chronic pain and responds poorly to the clinically available therapies. Studies from animal models of neuropathic pain have led to understanding of its pathobiology which includes complex interrelated pathways leading to peripheral and central neuronal sensitization. Advancements in the elucidation of neuropathic pain mechanisms have revealed a number of key targets that have been hypothesized to modulate clinical status. The present review discusses these therapeutic targets including noradrenaline and 5-HT reuptake inhibitors; sodium, calcium and potassium channels; inhibitory and excitatory neurotransmitters; neuropeptides including bradykinin, tachykinin, cholecystokinin, neuropeptide Y, vasoactive intestinal peptide, and CGRP; pro-inflammatory cytokines; MAP kinases; PPAR γ; Na(+)/Ca(2+) exchanger; nitric oxide; purinergic receptors; neuronal nicotinic receptors; cation-dependent chloride transporters; oxidative stress; matrix metalloproteinase and plasminogen activators; growth factors; transient receptor potential (TRP) channels; endocannabinoids; histamine receptors; dopamine; sigma receptors, beta adrenergic receptors, endothelins, and D-amino acid oxidase. The exploitation of these targets may provide effective therapeutic agents for the management of peripheral nerve injury-induced neuropathic pain.

Spinal Botulinum Neurotoxin B: Effects on Afferent Transmitter Release and Nociceptive Processing

Botulinum neurotoxin B (BoNT-B) mediates proteolytic cleavage of VAMP I/II (synaptobrevins I/II), which prevents vesicle-membrane fusion and blocks neurotransmitter release. In the present study, we investigated the effects of BoNT-B on neurotransmitter release in vivo from spinal primary afferent sensory fibers and the effects of this blockade on nociception. With intrathecally (IT) delivered BoNT-B in C57B/l6 mice, we characterized the effects of such block on the release of substance P (SP) from spinal afferent nociceptors (as measured by neurokinin-1 receptor, NK1-R, internalization), spinal neuronal activation (as indicated by spinal C-Fos expression) and nociceptive behavior after intraplantar (IPLT) formalin. In addition, we investigated the effect of IT BoNT-B on spinal nerve ligation-induced tactile allodynia. A single percutaneous IT injection of BoNT-B 0.5 U at 2 or 5 days prior to IPLT formalin reduced NK1-R internalization and C-Fos expression. These effects correlated with BoNT-B cleavage of VAMPI/II protein in tissue lysate. IT BoNT-B also produced a corresponding reduction in phase 2 of formalin-evoked flinching behavior for over 30 days after IT injection. In mice with spinal nerve ligation (SNL), tactile allodynia was observed, which was attenuated by IT BoNT-B 0.5 U over the next 15 days, as compared to vehicle animals. These effects were observed without effects upon motor function. The specificity of the IT BoNT-B effect is indicated by: i) IT co-injection of BoNT-B and anti-BoNTB antibody prevented effects on SP release, and ii) IT BoNT-B 50 U in the Sprague Dawley rats showed no effect on formalin-evoked flinching or SNL-induced tactile allodynia, which is consistent with rat resistance to BoNT-B. IT BoNT-B blocks transmitter release from spinal primary afferents, and attenuates inflammatory nociceptive response and spinal nerve injury-induced neuropathic pain, in the absence of motor impairment. These observations provide an initial assessment of the ability of IT BoNT-B to regulate spinal nociceptive processing.

Changes in expression of Nogo-A protein in dorsal root ganglion and spinal dorsal horn in a rat model of neuropathic pain

Objective To investigate the changes in the expression of Nogo-A protein in the dorsal root ganglion (DRG) and spinal dorsal horn in a rat model of neuropathic pain (NP) .Methods Seventy-two male SD rats weighing 250-300 g were randomly divided into 3 groups ( n = 24 each) : control group (group C) , sham operation group (group S) and NP group. NP was induced by ligation and severance of tibial and common fibular nerves according to the technique described by Isabelle et al. The mechanical withdrawal threshold (MWT) to von Frey filament stimulation was measured at 1, 7, 14 and 21 days after ligation. Six rats in each group were randomly selected at each time point and sacrificed (3 for determination of Nogo-A protein expression by immunofluorescence, 3 for determination of Nogo-A protein expression by Western blot) . The L5 DRG and L4,5 segment of spinal cord on the injured side were removed for determination of Nogo-A protein expression by immunofluorescence and Western blot. Results Compared with the groups C and S, MWT was significantly decreased at 7, 14 and 21 days after ligation, the expression of Nogo-A protein in the DRG was down-regulated at 7 and 14 days after ligation and the expression of Nogo-A protein in the spinal dorsal horn was up-regulated at 14 and 21 days after ligation ( P ＜0.05) .Conclusion The Nogo-A protein in the DRG and spinal dorsal horn may play an important role in peripheral nerve injury-induced NP in rats. Key words: Neuralgia; Spinal cord; Ganglia, spinal; Nogo-A protein

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

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.

Statins alleviate experimental nerve injury-induced neuropathic pain

The statins are a well-established class of drugs that lower plasma cholesterol levels by inhibiting HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase. They are widely used for the treatment of hypercholesterolemia and for the prevention of coronary heart disease. Recent studies suggest that statins have anti-inflammatory effects beyond their lipid-lowering properties. We sought to investigate whether statins could affect neuropathic pain by mediating nerve injury-associated inflammatory responses. The effects of hydrophilic rosuvastatin and lipophilic simvastatin were examined in the mouse partial sciatic nerve ligation model. Systemic daily administration of either statin from days 0 to 14 completely prevented the development of mechanical allodynia and thermal hyperalgesia. When administered from days 8 to 14 after injury, both statins dose-dependently reduced established hypersensitivity. After treatment, the effects of the statins were washed out within 2 to 7 days, depending on dose. Effects of both statins in alleviating mechanical allodynia were further confirmed in a different injury-associated neuropathic pain model, mental nerve chronic constriction, in rats. Both statins were able to abolish interleukin-1β expression in sciatic nerve triggered by nerve ligation. Additionally, quantitative analysis with Iba-1 and glial fibrillary acid protein immunoreactivity demonstrated that rosuvastatin and simvastatin significantly reduced the spinal microglial and astrocyte activation produced by sciatic nerve injury. The increase of interleukin-1β mRNA in the ipsilateral side of spinal cords was also reduced by the treatment of either statin. We identified a potential new application of statins in the treatment of neuropathic pain. The pain-alleviating effects of statins are likely attributable to their immunomodulatory effects. Statins, which are typically used to reduce blood cholesterol levels, could potentially have beneficial effects in the treatment of neuropathic pain.

P38/MAPK Inhibitor Modulates the Expression of Dorsal Horn GABA(B) Receptors in the Spinal Nerve Ligation Model of Neuropathic Pain

Background: Neuropathic pain is one of the most challenging clinical problems due to a lack of understanding the mechanisms. Recent studies have suggested that activated microglia in spinal cord may play a vital role in nerve injury-induced neuropathic pain, but the exact mechanisms have not been fully determined. Methods: First, we investigated the changes of dorsal horn GABA(B) receptor 1 (R1) expression in spinal nerve ligation rats. Second, we explored whether activated microglia contributed to such neuron changes by intrathecal administration of the p38 inhibitor, SB203580. Results: In this study, we found a dynamic change of GABA(B)R1a protein expression after spinal nerve ligation, and the peripheral nerve injury-induced downregulation of GABA(B)R1a expression in the spinal dorsal horn could be prevented by intrathecal administration of a p38/MAPK inhibitor SB203580. Conclusions: Our results provide valuable information for a better understanding of neuropathic pain and may contribute to developing effective treatments in future studies.

Role of different brain areas in peripheral nerve injury-induced neuropathic pain

Neuropathic pain has been described as the “most terrible of all tortures which a nerve wound may inflict” and arises as a consequence of nerve injury either of the peripheral or central nervous system. Following peripheral nerve injury, a cascade of events in the primary afferents leads to peripheral sensitization resulting in spontaneous nociceptor activity, decreased threshold and increased response to supra-threshold stimuli. A series of molecular changes in spinal cord and brain centers are associated with central sensitization which is responsible for the pain to non-injured extra-territory regions (extraterritorial pain) and contralateral parts (mirror-image pain). The peripheral nerve injury has been reported to induce neuroplastic changes in different brain regions including the anterior cingulate cortex, insular cortex, ventrolateral orbitofrontal area, amygdala, striatum, thalamus, hypothalamus, rostral ventromedial medulla, periaqueductal gray, pons (locus coeruleus), red nucleus, and medulla oblongata. The present review article discusses the involvement of these different brain areas in the development of peripheral nerve injury-induced neuropathic pain.

Toll-Like Receptor 2 Contributes to Chemokine Gene Expression and Macrophage Infiltration in the Dorsal Root Ganglia after Peripheral Nerve Injury

BackgroundWe have previously reported that nerve injury-induced neuropathic pain is attenuated in toll-like receptor 2 (TLR2) knock-out mice. In these mice, inflammatory gene expression and spinal cord microglia actvation is compromised, whereas the effects in the dorsal root ganglia (DRG) have not been tested. In this study, we investigated the role of TLR2 in inflammatory responses in the DRG after peripheral nerve injury.ResultsL5 spinal nerve transection injury induced the expression of macrophage-attracting chemokines such as CCL2/MCP-1 and CCL3/MIP-1 and subsequent macrophage infiltration in the DRG of wild-type mice. In TLR2 knock-out mice, however, the induction of chemokine expression and macrophage infiltration following nerve injury were markedly reduced. Similarly, the induction of IL-1β and TNF-α expression in the DRG by spinal nerve injury was ameliorated in TLR2 knock-out mice. The reduced inflammatory response in the DRG was accompanied by attenuation of nerve injury-induced spontaneous pain hypersensitivity in TLR2 knock-out mice.ConclusionsOur data show that TLR2 contributes to nerve injury-induced proinflammatory chemokine/cytokine gene expression and macrophage infiltration in the DRG, which may have relevance in the reduced pain hypersensitivity in TLR2 knock-out mice after spinal nerve injury.

Preserved Acute Pain and Impaired Neuropathic Pain in Mice Lacking Protein Interacting with C Kinase 1

Protein interacting with C Kinase 1 (PICK1), a PDZ domain-containing scaffolding protein, interacts with multiple different proteins in the mammalian nervous system and is believed to play important roles in diverse physiological and pathological conditions. In this study, we report that PICK1 is expressed in neurons of the dorsal root ganglion (DRG) and spinal cord dorsal horn, two major pain-related regions. PICK1 was present in approximately 29.7% of DRG neurons, most of which were small-less than 750 μm2 in cross-sectional area. Some of these PICK1-positive cells co-labeled with isolectin B4 or calcitonin-gene-related peptide. In the dorsal horn, PICK1 immunoreactivity was concentrated in the superficial dorsal horn, where it was prominent in the postsynaptic density, axons, and dendrites. Targeted disruption of PICK1 gene did not affect basal paw withdrawal responses to acute noxious thermal and mechanical stimuli or locomotor reflex activity, but it completely blocked the induction of peripheral nerve injury-induced mechanical and thermal pain hypersensitivities. PICK1 appears to be required for peripheral nerve injury-induced neuropathic pain development and to be a potential biochemical target for treating this disorder.

Non-beneficial effects of rosiglitazone in oxaliplatin-induced cold hyperalgesia in rats

Background: Studies have suggested the ameliorative potential of PPAR g agonist in attenuating the nerve injury-induced neuropathic pain. However, their role in chemotherapy-induced neuropathic pain is not explored yet. Aims: To investigate the potential of rosiglitazone, a PPAR g agonist, in oxaliplatin-induced cold hyperalgesia in rats. Settings and Design: All animals were divided in nine groups and single administration of oxaliplatin (6 mg/kg ip ) used for induction of neuropathy. Material and Methods : The pinprick, cold immersion, hot plate and hot immersion tests were performed to assess the degree of mechanical hyperalgesia, cold hyperalgesia, heat hyperalgesia, and heat allodynia, respectively. The levels of thiobarbituric acid reactive species (TBARS) and reduced glutathione (GSH) were measured as an index of oxidative stress. The myeloperoxidase (MPO) activity (a specific marker of inflammation) and calcium levels were also determined. Statistical analysis: Two-way analysis of variance (ANOVA) followed by Bonferroni's post test for behavioral assessment and one-way ANOVA followed by Tukey's multiple range tests for biochemical assessment were performed. Results: Single administration of oxaliplatin resulted in significant development of cold hyperalgesia without altering the nociceptive threshold for mechanical and heat stimuli. Furthermore, oxaliplatin increased the oxidative stress and decreased calcium levels without affecting inflammation. Treatment with rosiglitazone (2.5, 5, and 10 mg/kg po ) for 11 days did not modulate oxaliplatin-induced cold hyperalgesia. Moreover, rosiglitazone did not modulate oxaliplatin-induced biochemical changes. Conclusions: PPAR g agonists are ineffective in attenuating the state of cold hyperalgesia during oxaliplatin administration.

VGLUT2 expression in primary afferent neurons is essential for normal acute pain and injury-induced heat hypersensitivity

Dorsal root ganglia (DRG) neurons, including the nociceptors that detect painful thermal, mechanical, and chemical stimuli, transmit information to spinal cord neurons via glutamatergic and peptidergic neurotransmitters. However, the specific contribution of glutamate to pain generated by distinct sensory modalities or injuries is not known. Here we generated mice in which the vesicular glutamate transporter 2 (VGLUT2) is ablated selectively from DRG neurons. We report that conditional knockout (cKO) of the Slc17a6 gene encoding VGLUT2 from the great majority of nociceptors profoundly decreased VGLUT2 mRNA and protein in these neurons, and reduced firing of lamina I spinal cord neurons in response to noxious heat and mechanical stimulation. In behavioral assays, cKO mice showed decreased responsiveness to acute noxious heat, mechanical, and chemical (capsaicin) stimuli, but responded normally to cold stimulation and in the formalin test. Strikingly, although tissue injury-induced heat hyperalgesia was lost in the cKO mice, mechanical hypersensitivity developed normally. In a model of nerve injury-induced neuropathic pain, the magnitude of heat hypersensitivity was diminished in cKO mice, but both the mechanical allodynia and the microgliosis generated by nerve injury were intact. These findings suggest that VGLUT2 expression in nociceptors is essential for normal perception of acute pain and heat hyperalgesia, and that heat and mechanical hypersensitivity induced by peripheral injury rely on distinct (VGLUT2 dependent and VGLUT2 independent, respectively) primary afferent mechanisms and pathways.

The role of TLR2 in nerve injury‐induced neuropathic pain is essentially mediated through macrophages in peripheral inflammatory response

Activation of macrophages/microglia via toll-like receptors (TLRs) plays an important role in inflammation and host defense against pathogens. Pathogen-associated molecular patterns bind TLRs, thereby triggering NF-κB signaling and production of proinflammatory cytokines. Recent data suggest that nonpathogenic molecules resulting from trauma can also trigger inflammation via TLRs. We sought to determine whether peripheral nerve injury could induce the expression of TLR2 on the site of injury-damaged nerves and/or in the central nervous system and to investigate whether TLR2 is necessary for the development of nerve injury-induced neuropathic pain. We observed a significant increase in TLR2, IκB-α, and TNF-α mRNAs in damaged nerves. Increased inflammation-related molecules were found essentially on ED1(+) macrophages. Expression of both IκB-α and TNF-α in peripheral injured nerves was reduced in TLR2 deficient mice where the recruitment of ED1(+) cells is significantly impaired. Although after peripheral nerve injury, spinal microglia became highly activated showing an increase in Iba-1 immunoreactivity and an enlargement of their cell bodies, neither TLR2 mRNA nor IκB-α mRNA was detected in activated microglia. Nerve injury-evoked spinal microglial activation was not significantly altered in TLR2 KO mice. Paw withdrawal threshold and latency in response to mechanical and heat stimuli, respectively, decreased shortly after nerve lesion in wild type mice. In TLR2 KO mice, nerve injury-induced thermal hyperalgesia was completely abolished contrary to that seen in wild-type mice, whereas mechanical allodynia was partially reduced. We suggest that TLR2 is necessary for the development of neuropathic pain and its contribution is more important in thermal hypersensitivity than that of mechanical allodynia.

A novel mechanism of inhibition of high-voltage activated calcium channels by α-conotoxins contributes to relief of nerve injury-induced neuropathic pain

α-Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing α3-subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain-relieving actions of α-conotoxins might be mediated by either α9α10 nAChRs or a novel GABAB receptor-mediated inhibition of N-type calcium channels. Here we establish that three α-conotoxins, Vc1.1, AuIB and MII have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits α9α10 nAChRs and GABAB/Ca2+ channels but weakly blocks α3β2 and α3β4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pre-treatment with the GABAB receptor antagonist, SCH50911. α-Conotoxin AuIB, a weak α3β4 nAChR antagonist, inhibited GABAB/Ca2+ channels but did not act on α9α10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABAB receptor-dependent inhibition of N-type Ca2+ channels can mediate the sustained anti-allodynic actions of some α-conotoxins. However, MII, a potent α3β2 nAChR antagonist but inactive on α9α10 and α3β4 nAChRs and GABAB/Ca2+ channels, was demonstrated to have short-acting anti-allodynic action. This suggests that α3β2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of α9α10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that α-conotoxins selective for GABAB receptor-dependent inhibition of N-type Ca2+ channels relieve allodynia, and could therefore be developed to manage chronic pain.

Microglial activation mediates de novo lysophosphatidic acid production in a model of neuropathic pain

We recently demonstrated that de novo lysophosphatidic acid (LPA) production in the spinal cord occurs in the early phase after nerve injury or LPA injection, and underlies the peripheral mechanisms of neuropathic pain. In this study, we examined the possible involvement of spinal cord microglia in such LPA-mediated functions. Intrathecal LPA injection rapidly increased the gene expression of CD11b and protein expression of phosphor-p38, accompanied by a morphological change of microglia from a ramified to amoeboid shape. Although early treatment with minocycline significantly inhibited LPA-induced neuropathic pain-like behavior and microglial activation, late treatment did not. Early treatment with minocycline also blocked LPA-evoked de novo LPA production and the increased activation of cytosolic phospholipase A(2), an LPA synthesis-related enzyme. Similar results were observed when the sciatic nerve was partially injured: early, but not late, treatment with minocycline significantly inhibited the injury-induced neuropathic pain, microglial activation, de novo LPA production and the underlying increased activation of cytosolic phospholipase A(2) as well as calcium-independent phospholipase A(2), another LPA synthesis-related enzyme. These findings suggest that the early phase of microglial activation is involved in de novo LPA production, and that this underlies the initial mechanisms of nerve injury-induced neuropathic pain.

CC-chemokine MIP-1α in the spinal cord contributes to nerve injury-induced neuropathic pain

We investigated the involvement of spinal macrophage inflammatory protein-1α (MIP-1α), an inflammatory chemokine, in partial sciatic nerve ligation (PSL)-induced neuropathic pain in mice. PSL increased MIP-1α mRNA levels as well as levels of the MIP-1α receptor, CCR1, but not CCR5 in the spinal dorsal horn. PSL-induced tactile allodynia and thermal hyperalgesia were prevented by intrathecal (i.t.) injection of a neutralizing antibody of MIP-1α (2 ng). Recombinant MIP-1α (10 pmol, i.t.) elicited long-lasting tactile allodynia and thermal hyperalgesia in naïve mice. These results suggest that peripheral nerve injury elicits the up-regulation of spinal MIP-1α and CCR1 to participate in neuropathic pain.

NADPH oxidase 2-derived reactive oxygen species in spinal cord microglia contribute to peripheral nerve injury-induced neuropathic pain

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain.

Intrathecal Infusion of Pyrrolidine Dithiocarbamate for the Prevention and Reversal of Neuropathic Pain in Rats Using a Sciatic Chronic Constriction Injury Model

Recent studies have suggested that nuclear factor κB (NF-κB) may play a role in mediating nerve injury-induced neuropathic pain. Here, we examined the effects of intrathecal pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, on the development of neuropathic pain, spinal microglial activation, and CX3CR1 expression induced by sciatic chronic constriction injury (CCI) model in rats. Under chloral hydrate anesthesia, male Sprague-Dawley rats (300-350 g) fitted with intrathecal catheters underwent either sciatic CCI or sham surgery. Intrathecal saline or PDTC (100 or 1000 pmol/d) was infused 1 day before or 3 days after CCI (n = 8). The rat hind-paw withdrawal threshold to mechanical stimuli and withdrawal latency to radiant heat were determined before surgery and from days 1 to 7 after CCI. Spinal microglial activation was evaluated with OX-42 immunoreactivity, and spinal CX3CR1 expression was assessed by Western blotting. Chronic constriction injury induced mechanical allodynia and thermal hyperalgesia and microglial activation as demonstrated by OX-42 expression. Whereas it had no apparent effect on spinal cord histology, intrathecal administration of PDTC prevented the development of the mechanical and thermal hyperalgesia and inhibited nerve injury-induced microglial activation and spinal CX3CR1 expression. In this study, we have shown the protective effect of intrathecal PDTC on the development of nociceptive behaviors induced by CCI in rats. The activation of NF-κB pathway may contribute to spinal microglial activation and CX3CR1 up-regulation.

Calpain‐mediated down‐regulation of myelin‐associated glycoprotein in lysophosphatidic acid‐induced neuropathic pain

Lysophosphatidic acid receptor (LPA(1)) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal injection of LPA evoked a dose- and time-dependent down-regulation of myelin-associated glycoprotein (MAG) in the DR through LPA(1) receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA(1) receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund's adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR.

Decrease in the descending inhibitory 5-HT system in rats with spinal nerve ligation

The descending serotonergic (5-HT) system is shown to be plastically altered under pathological conditions such as inflammation or peripheral nerve lesion. Although much evidence indicates that the potentiation of descending facilitatory 5-HT pathways may contribute to the development of chronic pain, the inhibition of descending inhibitory 5-HT system may be functionally more important to the development of central sensitization and neuropathic pain. In the present study, we observed that the inhibitory effects of 5-HT and its receptor agonists including 1A, 1B, 3, 4, and probably 2C receptor agonists, on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in the spinal cord decreased significantly following spinal nerve ligation (SNL). Furthermore, we found that the antagonistic effects of 5-HT 1B, 2C, 3, and 4 receptor antagonists on the 5-HT-induced inhibition of C-fiber responses of WDR neurons were also attenuated after SNL. In consistent with these observations, we also found an obvious decrease in the content of 5-HT and 5-HIAA, and a marked increase in the turnover rate of 5-HT (5-HIAA/5-HT) in the ipsilateral dorsal half of the lumbar spinal cord after SNL. These data indicate that a loss or decrease in the descending inhibitory 5-HT system upon the spinal processing of nociceptive information appears to occur following spinal nerve injury, and this kind of decrease in the descending inhibitory 5-HT system is proposed to be involved in the development of central sensitization and ultimately to the nerve injury-induced neuropathic pain.