Shiping Zou

Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain.

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-1β (IL-1β), 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-1β was selectively localized to astrocytes but not to microglia and neurons. Local anesthesia of the masseter nerve prevented the increase in GFAP and IL-1β after inflammation, and substance P, a prototype neurotransmitter of primary afferents, induced similar increases in GFAP and IL-1β, which was blocked by a nitric oxide synthase inhibitor NG-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-1β 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-1β induction, and the coupling of NMDA receptor phosphorylation through IL-1 receptor signaling.

Supraspinal Glial-Neuronal Interactions Contribute to Descending Pain Facilitation

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-α (TNF-α) and interleukin-1β (IL-1β) 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-α and IL-1β, 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-α and IL-1β in the RVM significantly reduced CCI-induced behavioral hypersensitivity and attenuated NR1 phosphorylation. Finally, intra-RVM administration of recombinant TNF-α or IL-1β upregulated NR1 phosphorylation and caused a reversible and NMDAR-dependent allodynia in normal rats, further suggesting that TNF-α and IL-1β 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.

Group I Metabotropic Glutamate Receptor NMDA Receptor Coupling and Signaling Cascade Mediate Spinal Dorsal Horn NMDA Receptor 2B Tyrosine Phosphorylation Associated with Inflammatory Hyperalgesia

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 Freunds 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.

Supraspinal brain-derived neurotrophic factor signaling : A novel mechanism for descending pain facilitation

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 IP3, 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.

Molecular Depletion of Descending Serotonin Unmasks Its Novel Facilitatory Role in the Development of Persistent Pain

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 μ- or κ-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.

Inhibition of class II histone deacetylases in the spinal cord attenuates inflammatory hyperalgesia

BackgroundSeveral 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 Freunds adjuvant (CFA).ResultsWe 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.ConclusionsOur 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.

Orofacial Deep and Cutaneous Tissue Inflammation and Trigeminal Neuronal Activation

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.

Differential involvement of trigeminal transition zone and laminated subnucleus caudalis in orofacial deep and cutaneous hyperalgesia: the effects of interleukin-10 and glial inhibitors

BackgroundIn 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.ResultsDeep or cutaneous inflammatory hyperalgesia, assessed with von Frey filaments, was induced in rats by injecting complete Freunds 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 μg), and minocycline (0.1-1 μg) 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.ConclusionThese 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.

Bone Marrow Stromal Cells Produce Long‐Term Pain Relief in Rat Models of Persistent Pain

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. STEM CELLS 2011;29:1294–1303

Selective upregulation of the flip-flop splice variants of AMPA receptor subunits in the rat spinal cord after hindpaw inflammation.

Glutamate receptors are involved in spinal nociceptive transmission and the development of persistent inflammatory hyperalgesia. It is unclear, however, whether there are changes in glutamate receptor gene expression associated with tissue injury. In the present study, we used reverse transcription-polymerase chain reaction (RT-PCR) to examine the modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor gene expression in the rat spinal cord by inflammation. Inflammation was introduced into the hindpaw by intraplantar injection of 0.2 ml of complete Freunds adjuvant (CFA). At 2 h-14 d after inflammation, total RNAs from L4,5 spinal cord were used for RT-PCR with primers targeted at eight flip-flop splice variants of the AMPA receptor subunits. It was found that the GluR1-flop mRNA was up-regulated at 2 h-5 h (P<0.05), down-regulated at 3 d (P=0.05), and returned to control levels at 7 d following inflammation. The GluR2-flip and GluR3-flop mRNAs were up-regulated at 5 h-1 d (P<0.05) and returned to control levels at 3 d after inflammation. The GluR1-flip mRNA was not detected in the samples and the mRNAs for other splice variants did not exhibit significant changes. Immunocytochemical analysis of GluR1 and GluR2 subunits indicate that the protein translation products of these subunits were also increased in the spinal cord. These results demonstrate an increased expression of AMPA receptor subunits that correlates with the acute phase of CFA-induced inflammation and hyperalgesia. Selective changes in the expression of the flip-flop splice variants of the AMPA receptor suggest a reorganization of the composition of the AMPA receptor complex and its involvement in the development of inflammatory hyperalgesia.