[show abstract][hide abstract] ABSTRACT: The orofacial region is a major focus of chronic neuropathic pain conditions characterized by primary hyperalgesia at the site of injury and secondary hyperalgesia outside the injured zone. We have used a rat model of injury to the maxillary branch (V2) of the trigeminal nerve to produce constant and long-lasting primary hyperalgesia in the V2 territory and secondary hyperalgesia in territories innervated by the mandibular branch (V3). Our findings indicate that the induction of primary and secondary hyperalgesia depended on peripheral input from the injured nerve. In contrast, the maintenance of secondary hyperalgesia depended on central mechanisms. The centralization of the secondary hyperalgesia involved descending 5-HT drive from the rostral ventromedial medulla and the contribution of 5-HT3 receptors in the trigeminal nucleus caudalis (Vc), the homolog of the spinal dorsal horn. Electrophysiological studies further indicate that after nerve injury spontaneous responses and enhanced poststimulus discharges in Vc nociresponsive neurons were time-dependent on descending 5-HT drive and peripheral input. The induction phase of secondary hyperalgesia involved central sensitization mechanisms in Vc neurons that were dependent on peripheral input, whereas the maintenance phase of secondary hyperalgesia involved central sensitization in Vc neurons conducted by a delayed descending 5-HT drive and a persistence of peripheral inputs. Our results are the first to show that the maintenance of secondary hyperalgesia and underlying central sensitization associated with persistent pain depend on a transition to supraspinal mechanisms involving the serotonin system in rostral ventromedial medulla-dorsal horn circuits.
Journal of Neuroscience 03/2013; 33(12):5152-61. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies indicate that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT(3) receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT(3) receptor and its contribution to facilitation of pain remain unclear. In the present study, activation of spinal 5-HT(3) receptor by intrathecal injection of a selective 5-HT(3) receptor agonist, SR57227, induced spinal glial hyperactivity, neuronal hyperexcitability, and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via chemokine fractalkine, microglia to astrocyte signaling via the cytokine IL-18, astrocyte to neuronal signaling by IL-1β, and enhanced activation of GluN (NMDA) receptors in the spinal dorsal horn. In addition, exogenous brain-derived neurotrophic factor-induced descending pain facilitation was accompanied by upregulation of CD11b and GFAP expression in the spinal dorsal horn after microinjection in the RVM, and these events were significantly prevented by functional blockade of spinal 5-HT(3) receptors. Enhanced expression of spinal CD11b and GFAP after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. Thus, these findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neuron and glia.
Journal of Neuroscience 09/2011; 31(36):12823-36. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chronic pain conditions are difficult to treat and are major health problems. Bone marrow stromal cells (BMSCs) have generated considerable interest as a candidate for cell-based therapy. BMSCs are readily accessible and are easy to isolate and expand ex vivo. Clinical studies show that direct injection of BMSCs does not produce unwanted side effects and is well tolerated and safe. Here, we show that a single systemic (intravenous) or local injection (into the lesion site) of rat primary BMSCs reversed pain hypersensitivity in rats after injury and that the effect lasted until the conclusion of the study at 22 weeks. The pain hypersensitivity was rekindled by naloxone hydrochloride, an opioid receptor antagonist that acts peripherally and centrally, when tested at 1-5 weeks after BMSC infusion. In contrast, naloxone methiodide, a peripherally acting opioid receptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC treatment. Focal downregulation of brainstem mu opioid receptors by RNA interference (RNAi) reversed the effect of BMSCs, when RNAi was introduced at 5- but not 1-week after BMSC transplantation. Thus, BMSCs produced long-term relief of pain and this effect involved activation of peripheral and central opioid receptors in distinct time domains. The findings prompt studies to elucidate the cellular mechanisms of the BMSC-induced pain relieving effect and translate these observations into clinical settings.
[show abstract][hide abstract] ABSTRACT: Recent studies indicate that persistent pain after tissue or nerve injury is accompanied by an enhanced net descending facilitatory drive that contributes to an amplification and spread of pain. Although 5-HT-containing neurons in the rostral ventromedial medulla (RVM) provide the major descending serotonergic projection to the spinal cord, it is not clear whether the neurotransmitter 5-HT itself released from RVM-spinal neurons contributes to descending pain modulation. In the present study, we determined the role of the descending 5-HT in rat nocifensive behaviors after persistent pain by selectively depleting functional phenotypes of 5-HT in RVM neurons with regional shRNA interference (RNAi) of tryptophan hydroxylase-2 (Tph-2), the rate-limiting enzyme in the synthesis of neuronal 5-HT. Compared to negative control shRNA, Tph-2 shRNA induced significantly prolonged downregulation of Tph-2 in the RVM and 5-HT in spinal dorsal horn. The 5-HT-depleted rats showed normal pain sensitivity in responses to acute noxious stimulation. However, the same RNAi treatment attenuated formalin-induced spontaneous nocifensive responses and tissue or nerve injury-induced allodynia and hyperalgesia. Furthermore, in control shRNA-treated animals, intra-RVM microinjection of brain-derived neurotrophic factor produced a reversible hyperalgesia, which was completely prevented by Tph-2 RNAi pretreatment. Descending inhibition induced by intra-RVM electrical stimulation, but not microinjection of the mu- or kappa-opioid receptor agonists in control shRNA-treated animals was eliminated in 5-HT-depleted rats. These results indicate that the descending 5-HT from the RVM is an important contributor to pain facilitation during the development of persistent pain, and may not mediate opioid-induced descending inhibition in acute pain.
Journal of Neuroscience 06/2010; 30(25):8624-36. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several classes of histone deacetylases (HDACs) are expressed in the spinal cord that is a critical structure of the nociceptive pathway. HDAC-regulated histone acetylation is an important component of chromatin remodeling leading to epigenetic regulation of gene transcription. To understand the role of histone acetylation in epigenetic regulation of pathological pain, we have studied the impact of different classes of HDACs in the spinal cord on inflammatory hyperalgesia induced by complete Freund's adjuvant (CFA).
We intrathecally applied inhibitors specific to different classes of HDACs and evaluated their impact on inflammatory hyperalgesia. Pre-injected inhibitors targeting class I as well as II (SAHA, TSA, LAQ824) or IIa (VPA, 4-PB) HDACs significantly delayed the thermal hyperalgesia induced by unilateral CFA injection in the hindpaw. Existing hyperalgesia induced by CFA was also attenuated by the HDAC inhibitors (HDACIs). In contrast, these inhibitors did not interfere with the thermal response either in naïve animals, or on the contralateral side of inflamed animals. Interestingly, MS-275 that specifically inhibits class I HDACs failed to alter the hyperalgesia although it increased histone 3 acetylation in the spinal cord as SAHA did. Using immunoblot analysis, we further found that the levels of class IIa HDAC members (HDAC4, 5, 7, 9) in the spinal dorsal horn were upregulated following CFA injection while those of class I HDAC members (HDAC1, 2, 3) remained stable or were slightly reduced.
Our data suggest that activity of class II HDACs in the spinal cord is critical to the induction and maintenance of inflammatory hyperalgesia induced by CFA, while activity of class I HDACs may be unnecessary. Comparison of the effects of HDACIs specific to class II and IIa as well as the expression pattern of different HDACs in the spinal cord in response to CFA suggests that the members of class IIa HDACs may be potential targets for attenuating persistent inflammatory pain.
[show abstract][hide abstract] ABSTRACT: A major subgroup of patients with temporomandibular joint (TMJ) disorders have masticatory muscle hypersensitivity. To study myofacial temporomandibular pain, a number of preclinical models have been developed to induce myogenic pain of the masseter muscle, one of the four muscles involved in mastication. The currently used models, however, generate pain that decreases over time and only lasts from hours to weeks and hence are not suitable for studying chronicity of the myogenic pain in TMJ disorders. Here we report a model of constant myogenic orofacial pain that lasts for months.
The model involves unilateral ligation of the tendon of the anterior superficial part of the rat masseter muscle (TASM). The ligation of the TASM was achieved with two chromic gut (4.0) ligatures via an intraoral approach. Nocifensive behavior of the rat was assessed by probing the skin site above the TASM with a series of von Frey filaments. The response frequencies were determined and an EF50 value, defined as the von Frey filament force that produces a 50% response frequency, was derived and used as a measure of mechanical sensitivity. Following TASM ligation, the EF50 of the injured side was significantly reduced and maintained throughout the 8-week observation period, suggesting the presence of mechanical hyperalgesia/allodynia. In sham-operated rats, the EF50 of the injured side was transiently reduced for about a week, likely due to injury produced by the surgery. Somatotopically relevant Fos protein expression was indentified in the subnucleus caudalis of the spinal trigeminal sensory complex. In the same region, persistent upregulation of NMDA receptor NR1 phosphorylation and protein expression and increased expression of glial markers glial fibrillary acidic protein (astroglia) and CD11b (microglia) were found. Morphine (0.4-8 mg/kg, s.c.) and duloxetine (0.4-20 mg/kg, i.p.), a selective serotonin-norepinephrine reuptake inhibitor, produced dose-dependent attenuation of hyperalgesia.
Ligation injury of the TASM in rats led to long-lasting and constant mechanical hypersensitivity of myogenic origin. The model will be particularly useful in studying the chronicity of myogenic pain TMJ disorders. The model can also be adapted to other regions of the body for studying pathology of painful tendinopathy seen in sports injury, muscle overuse, and rheumatoid arthritis.
[show abstract][hide abstract] ABSTRACT: In addition to caudal subnucleus caudalis (Vc) of the spinal trigeminal complex, recent studies indicate that the subnuclei interpolaris/caudalis (Vi/Vc) transition zone plays a unique role in processing deep orofacial nociceptive input. Studies also suggest that glia and inflammatory cytokines contribute to the development of persistent pain. By systematically comparing the effects of microinjection of the antiinflammatory cytokine interleukin (IL)-10 and two glial inhibitors, fluorocitrate and minocycline, we tested the hypothesis that there was a differential involvement of Vi/Vc and caudal Vc structures in deep and cutaneous orofacial pain.
Deep or cutaneous inflammatory hyperalgesia, assessed with von Frey filaments, was induced in rats by injecting complete Freund's adjuvant (CFA) into the masseter muscle or skin overlying the masseter, respectively. A unilateral injection of CFA into the masseter or skin induced ipsilateral hyperalgesia that started at 30 min, peaked at 1 d and lasted for 1-2 weeks. Secondary hyperalgesia on the contralateral site also developed in masseter-, but not skin-inflamed rats. Focal microinjection of IL-10 (0.006-1 ng), fluorocitrate (1 microg), and minocycline (0.1-1 microg) into the ventral Vi/Vc significantly attenuated masseter hyperalgesia bilaterally but without an effect on hyperalgesia after cutaneous inflammation. Injection of the same doses of these agents into the caudal Vc attenuated ipsilateral hyperalgesia after masseter and skin inflammation, but had no effect on contralateral hyperalgesia after masseter inflammation. Injection of CFA into the masseter produced significant increases in N-methyl-D-aspartate (NMDA) receptor NR1 serine 896 phosphorylation and glial fibrillary acidic protein (GFAP) levels, a marker of reactive astrocytes, in Vi/Vc and caudal Vc. In contrast, cutaneous inflammation only produced similar increases in the Vc.
These results support the hypothesis that the Vi/Vc transition zone is involved in deep orofacial injury and suggest that glial inhibition and interruption of the cytokine cascade after inflammation may provide pain relief.
[show abstract][hide abstract] ABSTRACT: Injection of endothelin-1 (ET-1) into the plantar rat hindpaw causes acute pain at high concentrations and tactile sensitization at low concentrations. The pro-nociceptive actions are driven through ET(A) receptors for both levels of [ET-1], but the ET(B) receptors are only pro-nociceptive for allodynia from low [ET-1] and anti-nociceptive for pain from high [ET-1]. The goal of the present work was to discriminate the roles of the ET receptors in the acute hyperalgesia from inflammation by complete Freund's adjuvant (CFA, 20 mg/paw) into the rat hindpaw. Selective antagonists were injected l0 min before and then together with CFA. An ET(A) receptor antagonist, BQ-123, reduced CFA-induced thermal hyperalgesia (by up to 50%), as did an ET(B) receptor antagonist, BQ-788 (by up to 66%). BQ-123 and BQ-788 also delayed the onset (by 1.5 - 2 h) but insignificantly reduced the maximum degree of CFA-induced allodynia (~10%). Surprisingly, an ET(B) receptor agonist, IRL-1620, also reduced maximum thermal hyperalgesia induced by CFA, suppressed peak allodynia and delayed its occurrence by ~ 3 h. The latter actions of IRL-1620 were reversed by co-administration of BQ-788, naloxone hydrochloride and the peripherally restricted opiate receptor antagonist naloxone methiodide, and by antiserum against β-endorphin. These findings demonstrate an important role for endogenous ET-1 in acute inflammatory pain and a dual action of ET(B) receptors, including a pro-algesic action along with the important activation of a local analgesic pathway, implying that at least two different ET(B) receptors contribute to modulation of inflammatory pain.
[show abstract][hide abstract] ABSTRACT: Spinal glial reaction and proinflammatory cytokine induction play an important role in the development of chronic pain states after tissue and nerve injury. The present study investigated the cellular and molecular mechanisms underlying descending facilitation of neuropathic pain with an emphasis on supraspinal glial-neuronal relationships. An early and transient reaction of microglia and prolonged reaction of astrocytes were found after chronic constriction injury (CCI) of the rat infraorbital nerve in the rostral ventromedial medulla (RVM), a major component of brainstem descending pain modulatory circuitry. There were prolonged elevations of cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) after CCI, and they were expressed in RVM astrocytes at 14 d after injury. Intra-RVM injection of microglial and astrocytic inhibitors attenuated mechanical hyperalgesia and allodynia at 3 and 14 d after CCI, respectively. Moreover, TNFR1 and IL-1R, receptors for TNF-alpha and IL-1beta, respectively, were expressed primarily in RVM neurons exhibiting immunoreactivity to the NMDA receptor (NMDAR) subunit NR1. CCI increased TNFR1 and IL-1R levels and NR1 phosphorylation in the RVM. Neutralization of endogenous TNF-alpha and IL-1beta in the RVM significantly reduced CCI-induced behavioral hypersensitivity and attenuated NR1 phosphorylation. Finally, intra-RVM administration of recombinant TNF-alpha or IL-1beta upregulated NR1 phosphorylation and caused a reversible and NMDAR-dependent allodynia in normal rats, further suggesting that TNF-alpha and IL-1beta couple glial hyperactivation with NMDAR function. These studies have addressed a novel contribution of supraspinal astrocytes and associated cytokines as well as central glial-neuronal interactions to the enhancement of descending facilitation of neuropathic pain.
Journal of Neuroscience 11/2008; 28(42):10482-95. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The emerging literature implicates a role for glia/cytokines in persistent pain. However, the mechanisms by which these non-neural elements contribute to CNS activity-dependent plasticity and pain are unclear. Using a trigeminal model of inflammatory hyperalgesia, here we provide evidence that demonstrates a mechanism by which glia interact with neurons, leading to activity-dependent plasticity and hyperalgesia. In response to masseter inflammation, there was an upregulation of glial fibrillary acidic proteins (GFAPs), a marker of astroglia, and interleukin-1beta (IL-1beta), a prototype proinflammatory cytokine, in the region of the trigeminal nucleus specifically related to the processing of deep orofacial input. The activated astroglia exhibited hypertrophy and an increased level of connexin 43, an astroglial gap junction protein. The upregulated IL-1beta was selectively localized to astrocytes but not to microglia and neurons. Local anesthesia of the masseter nerve prevented the increase in GFAP and IL-1beta after inflammation, and substance P, a prototype neurotransmitter of primary afferents, induced similar increases in GFAP and IL-1beta, which was blocked by a nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester. Injection of IL-1 receptor antagonist and fluorocitrate, a glial inhibitor, attenuated hyperalgesia and NMDA receptor phosphorylation after inflammation. In vitro application of IL-1beta induced NR1 phosphorylation, which was blocked by an IL-1 receptor antagonist, a PKC inhibitor (chelerythrine), an IP3 receptor inhibitor (2-aminoethoxydiphenylborate), and inhibitors of phospholipase C [1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione] and phospholipase A2 (arachidonyltrifluoromethyl ketone). These findings provide evidence of astroglial activation by tissue injury, concomitant IL-1beta induction, and the coupling of NMDA receptor phosphorylation through IL-1 receptor signaling.
Journal of Neuroscience 06/2007; 27(22):6006-18. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the adult mammalian brain, brain-derived neurotrophic factor (BDNF) is critically involved in long-term synaptic plasticity. Here, we show that supraspinal BDNF-tyrosine kinase receptor B (TrkB) signaling contributes to pain facilitation. We show that BDNF-containing neurons in the periaqueductal gray (PAG), the central structure for pain modulation, project to and release BDNF in the rostral ventromedial medulla (RVM), a relay between the PAG and spinal cord. BDNF in PAG and TrkB phosphorylation in RVM neurons are upregulated after inflammation. Intra-RVM sequestration of BDNF and knockdown of TrkB by RNA interference attenuate inflammatory pain. Microinjection of BDNF (10-100 fmol) into the RVM facilitates nociception, which is dependent on NMDA receptors (NMDARs). In vitro studies with RVM slices show that BDNF induces tyrosine phosphorylation of the NMDAR NR2A subunit in RVM via a signal transduction cascade involving IP(3), PKC, and Src. The supraspinal BDNF-TrkB signaling represents a previously unknown mechanism underlying the development of persistent pain. Our findings also caution that application of BDNF for recovery from CNS disorders could lead to undesirable central pain.
Journal of Neuroscience 02/2006; 26(1):126-37. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study was undertaken to evaluate the changes in cytokine levels in response to orofacial deep tissue inflammation. Inflammation was induced by injecting complete Freund's adjuvant (CFA, 0.05 ml 1:1 oil/saline suspension) into the masseter of the male Sprague-Dawley rat under brief halothane anesthesia. At 30 min, 5 h and 24 h after CFA injection (n = 3-4/time point), tissues were dissected from masseter and total proteins isolated. Rat Cytokine Antibody Array 1.1 (RayBiotech) coated with 19 specific cytokine antibodies were probed with protein samples and the relative cytokine levels were compared. Compared to saline-injected rats, there were significant increases (p < 0.05-0.01) in the levels of seven cytokines in the masseter tissue after CFA, including interleukin (IL)-1beta (5 h), IL-6 (5 h), tumor necrosis factor-alpha (5 h), monocyte chemoattractant protein-1 (5 h, 24 h), cytokine-induced neutrophil chemoattractant-2 and -3 (5 h, 24 h), and tissue inhibitor of metalloproteinase-1 (5 h, 24 h). All 19 cytokines were detected in the blood samples, but they did not show significant changes after inflammation. Masseter hyperalgesia and allodynia occurred at 30 min and persisted at 5-24 h after inflammation, as assessed by probing the skin above the masseter with von Frey filaments. The present results indicate selective localized cytokine responses to masseter inflammation. Although different cytokines exist in the blood, their levels did not mirror, nor did not appear to depend on, the local cytokine levels. The findings provide specific targets for further studying the involvement of cytokines in orofacial inflammation and hyperalgesia.
[show abstract][hide abstract] ABSTRACT: Hindpaw inflammation induces tyrosine phosphorylation (tyr-P) of the NMDA receptor (NMDAR) 2B (NR2B) subunit in the rat spinal dorsal horn that is closely related to the initiation and development of hyperalgesia. Here, we show that in rats with Freund's adjuvant-induced inflammation, the increased dorsal horn NR2B tyr-P is blocked by group I metabotropic glutamate receptor (mGluR) antagonists [7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP), by the Src inhibitor CGP 77675, but not by the MAP kinase inhibitor 2'-amino-3'-methoxyflavone. Analysis of the calcium pathways shows that the in vivo NR2B tyr-P is blocked by an IP3 receptor antagonist 2-aminoethoxydiphenylborate (2APB) but not by antagonists of ionotropic glutamate receptors and voltage-dependent calcium channels, suggesting that the NR2B tyr-P is dependent on intracellular calcium release. In a dorsal horn slice preparation, the group I (dihydroxyphenylglycine), but not group II [(2R,4R)-4-aminopyrrolidine-2,3-dicarboxylate] and III [L-AP 4 (L-(+)-2-amino-4-phosphonobutyric acid)], mGluR agonists, an IP3 receptor (D-IP3) agonist, and a PKC (PMA) activator, induces NR2B tyr-P similar to that seen in vivo after inflammation. Coimmunoprecipitation indicates that Shank, a postsynaptic density protein associated with mGluRs, formed a complex involving PSD-95 (postsynaptic density-95), NR2B, and Src in the spinal dorsal horn. Double immunofluorescence studies indicated that NR1 is colocalized with mGluR5 in dorsal horn neurons. mGluR5 also coimmunoprecipitates with NR2B. Finally, intrathecal pretreatment of CPCCOEt, MPEP, and 2APB attenuates inflammatory hyperalgesia. Thus, inflammation and mGluR-induced NR2B tyr-P share similar mechanisms. The group ImGluR-NMDAR coupling cascade leads to phosphorylation of the NMDAR and appears necessary for the initiation of spinal dorsal horn sensitization and behavioral hyperalgesia after inflammation.
Journal of Neuroscience 11/2004; 24(41):9161-73. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: We determined whether neural responses to inflammation and hyperalgesia involve activation of kainate receptors, a subgroup of glutamate receptors. Inflammation was introduced into the hind paw by intraplantar injection of complete Freund's adjuvant. The inflammation-induced thermal hyperalgesia was attenuated by intrathecal administration of a non-selective alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX), as well as by selective kainate receptor antagonists, 6,7,8,9-tetrohydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS-102) and 3S,4aR,6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid (LY382884). Reverse transcription-polymerase chain reaction (RT-PCR) indicated that the GluR5 and GluR6, but not the GluR7, KA1 and KA2 subunits, exhibited increased mRNA expression at 2 h to 3 days following inflammation (P<0.05). Western blot showed an increase in GluR6 protein levels (P<0.01) with a time course consistent with the changes in its mRNA levels. cDNA sequence and BbvI endonuclease digestion of the GluR6 PCR product revealed that the upregulated GluR6 mRNAs were predominantly the unedited form (Q). These results suggest that a selective upregulation of kainate receptor subunit expression contributes to inflammatory hyperalgesia.
European Journal of Pharmacology 10/2002; 452(3):309-18. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study examined the levels of NMDA receptor NR2 subunit tyrosine phosphorylation in a rat model of inflammation and correlated it with the development of inflammation and hyperalgesia. Hindpaw inflammation and hyperalgesia were induced by intraplantar injection of complete Freund's adjuvant. Proteins from the spinal cord (L4-L5) were immunoprecipitated with anti-NR2A or anti-NR2B antibodies and used for subsequent analysis using 4G-10, a specific anti-phosphotyrosine antibody. Compared with naive rats, there was a rapid and prolonged increase in tyrosine phosphorylation of the NR2B, but not NR2A, subunit after inflammation. The increase in NR2B tyrosine phosphorylation was dependent on primary afferent drive because (1) the phosphorylation correlated with the temporal profile of inflammation and hyperalgesia, (2) shorter-duration noxious stimulation produced a rapid and shorter-lasting increase in phosphorylation, and (3) local anesthetic block of the injected paw reversibly blocked inflammation-induced NR2B tyrosine phosphorylation and delayed hyperalgesia. The increase in NR2B tyrosine phosphorylation was abolished by intrathecal pretreatment with genistein, a tyrosine kinase inhibitor; PP2, an Src family tyrosine kinase inhibitor; AIDA, a group I metabotropic glutamate receptor antagonist; L733,060, an NK1 tachykinin receptor antagonist, and chelerythrine, a protein kinase C inhibitor. In addition, intrathecal PP2 delayed the onset of mechanical hyperalgesia and allodynia. These findings correlate in vivo NMDA receptor tyrosine phosphorylation with the development and maintenance of inflammatory hyperalgesia and suggest that signal transduction upstream to NR2B tyrosine phosphorylation involves G-protein-coupled receptors and PKC and Src family protein tyrosine kinases.
Journal of Neuroscience 08/2002; 22(14):6208-17. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: A rat model has been developed to characterize the responses of brainstem trigeminal neurons to orofacial deep and cutaneous tissue inflammation and hyperalgesia. Complete Freund’s adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. The TMJ and PO inflammation resulted in orofacial behavioral hyperalgesia and allodynia that peaked within 4–24 h and persisted for at least 2 weeks. Compared to cutaneous CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a selective upregulation of preprodynorphin mRNA in the trigeminal spinal complex, an enhanced medullary dorsal horn hyperexcitability, and a greater trigeminal Fos protein expression, a marker of neuronal activation. The Fos-LI induced by TMJ inflammation persisted longer, was more intense, particularly in the superficial laminae, and more widespread rostrocaudally. Thus, the inflammatory irritant produces a stronger effect in deep than in cutaneous orofacial tissue. As there is heavy innervation of the TMJ by unmyelinated nerve endings, a strong nociceptive primary afferent barrage is expected following inflammation. An increase in TMJ C-fiber input after inflammation and strong central neuronal activation may initiate central hyperexcitability and contribute to persistent pain associated with temporomandibular disorders. Since deep inputs may be more effective in inducing central neuronal excitation than cutaneous inputs, greater sensory disturbances may occur in pain conditions involving deep tissues than in those involving cutaneous tissues.