Xue-Jun Song

Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Here we show that Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repelled CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity was blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocked the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons.

WNT signaling underlies the pathogenesis of neuropathic pain in rodents

Treating neuropathic pain is a major clinical challenge, and the underlying mechanisms of neuropathic pain remain elusive. We hypothesized that neuropathic pain-inducing nerve injury may elicit neuronal alterations that recapitulate events that occur during development. Here, we report that WNT signaling, which is important in developmental processes of the nervous system, plays a critical role in neuropathic pain after sciatic nerve injury and bone cancer in rodents. Nerve injury and bone cancer caused a rapid-onset and long-lasting expression of WNTs, as well as activation of WNT/frizzled/β-catenin signaling in the primary sensory neurons, the spinal dorsal horn neurons, and astrocytes. Spinal blockade of WNT signaling pathways inhibited the production and persistence of neuropathic pain and the accompanying neurochemical alterations without affecting normal pain sensitivity and locomotor activity. WNT signaling activation stimulated production of the proinflammatory cytokines IL-18 and TNF-α and regulated the NR2B glutamate receptor and Ca2+-dependent signals through the β-catenin pathway in the spinal cord. These findings indicate a critical mechanism underlying the pathogenesis of neuropathic pain and suggest that targeting the WNT signaling pathway may be an effective approach for treating neuropathic pain, including bone cancer pain.

EphrinB-EphB receptor signaling contributes to neuropathic pain by regulating neural excitability and spinal synaptic plasticity in rats.

Abstract Bidirectional signaling between ephrins and Eph receptor tyrosine kinases was first found to play important roles during development, but recently has been implicated in synaptic plasticity and pain processing in the matured nervous system. We show that ephrinB‐EphB receptor signaling plays a critical role is induction and maintenance of neuropathic pain by regulating neural excitability and synaptic plasticity in the dorsal root ganglion (DRG) and the spinal dorsal horn (DH). Intrathecal application of blocking reagents for EphB‐receptors, EphB1‐Fc and EphB2‐Fc chimeras inhibits the induction and maintenance of nerve injury‐induced thermal hyperalgesia and mechanical allodynia. These blockers also prevent and suppress the nerve injury‐induced hyperexcitability of nociceptive small DRG neurons, sensitization of DH neurons and long‐term potentiation (LTP) of synapses between C fibers and DH neurons. In naïve, uninjured animals intrathecal administration of EphB‐receptor activators ephrinB1‐Fc and ephrinB2‐Fc, respectively, induces thermal hypersensitivity and lowers the threshold for LTP, while EphB1‐Fc prevents induction of the LTP. Western Blot analysis shows that nerve injury triggers an upregulation of the ephrinB1 and EphB1 receptor proteins in DRG and the spinal cord. These results indicate that, by regulating excitability of nociceptive‐related neurons in DRG and DH and the synaptic plasticity at the spinal level, ephrinB‐EphB receptor signaling contributes to neuropathic pain. This novel role for ephrinB‐EphB receptor signaling suggests that these molecules may be useful therapeutic targets for treating pain after nerve injury.

Blocking EphB1 Receptor Forward Signaling in Spinal Cord Relieves Bone Cancer Pain and Rescues Analgesic Effect of Morphine Treatment in Rodents

Treating bone cancer pain continues to be a clinical challenge and underlying mechanisms of bone cancer pain remain elusive. Here, we report that EphB1 receptor forward signaling in the spinal cord is critical to the development of bone cancer pain and morphine tolerance in treating bone cancer pain. Tibia bone cavity tumor cell implantation (TCI) produces bone cancer-related thermal hyperalgesia, mechanical allodynia, spontaneous and movement-evoked pain behaviors, and bone destruction. Production and persistence of these pain behaviors are well correlated with TCI-induced upregulation of EphB1 receptor and its ligand ephrinB2 in the dorsal horn and primary sensory neurons. Spinal administration of an EphB1 receptor blocking reagent EphB2-Fc prevents and reverses bone cancer pain behaviors and the associated induction of c-Fos and activation of astrocytes and microglial cells, NR1 and NR2B receptors, Src within the N-methyl-D-aspartate receptor complex, and the subsequent Ca(2+)-dependent signals. The exogenous ligand ephrinB2-Fc upregulates level of phosphorylation of NR1 and NR2B receptors depending on the activation of EphB1 receptor. Spinal administration of EphB2-Fc and ephrinB2-Fc induces downregulation of EphB1 and ephrinB2, respectively, accompanied with increased activity of matrix metalloproteinase (MMP)-2/9. Blocking MMP-2 or MMP-9 reverses EphB1-Fc treatment-induced downregulation of EphB1 receptor. In addition, spinal blocking or targeted mutation of EphB1 receptor reverses morphine tolerance in treating bone cancer pain in rats and defensive pain in mice. These findings show a critical mechanism underlying the pathogenesis of bone cancer pain and suggest a potential target for treating bone cancer pain and improving analgesic effect of morphine clinically.

Thiamine, pyridoxine, cyanocobalamin and their combination inhibit thermal, but not mechanical hyperalgesia in rats with primary sensory neuron injury.

&NA; Neuropathic pain after nerve injury is severe and intractable, and current drugs and nondrug therapies offer substantial pain relief to no more than half of affected patients. The present study investigated the analgesic roles of the B vitamins thiamine (B1), pyridoxine (B6) and cyanocobalamin (B12) in rats with neuropathic pain caused by spinal ganglia compression (CCD) or loose ligation of the sciatic nerve (CCI). Thermal hyperalgesia was determined by a significantly shortened latency of foot withdrawal to radiant heat, and mechanical hyperalgesia was determined by a significantly decreased threshold of foot withdrawal to von Frey filaments stimulation of the plantar surface of hindpaw. Results showed that (1) intraperitoneal injection of B1 (5, 10, 33 and 100 mg/kg), B6 (33 and100 mg/kg) or B12 (0.5 and 2 mg/kg) significantly reduced thermal hyperalgesia; (2) the combination of B1, B6 and B12 synergistically inhibited thermal hyperalgesia; (3) repetitive administration of vitamin B complex (containing B1/B6/B12 33/33/0.5 mg/kg, for 1 and 2 wk) produced long‐term inhibition of thermal hyperalgesia; and (4) B vitamins did not affect mechanical hyperalgesia or normal pain sensation, and exhibited similar effects on CCD and CCI induced‐hyperalgesia. The present studies demonstrate effects of B vitamins on pain and hyperalgesia following primary sensory neurons injury, and suggest the possible clinical utility of B vitamins in the treatment of neuropathic painful conditions following injury, inflammation, degeneration or other disorders in the nervous systems in human beings.

Somata of nerve-injured sensory neurons exhibit enhanced responses to inflammatory mediators.

The effects of inflammatory mediators in modulating the activity of nerve‐injured dorsal root ganglion (DRG) neurons were studied in rats in an in vitro nerve‐DRG preparation 2–4 weeks after a loose ligation of the sciatic nerve (chronic constriction injury, CCI). An inflammatory soup (IS) of bradykinin, serotonin, prostaglandin E2 and histamine (each 10−5 M, pH=7.4) was applied topically to the DRG. Evoked responses were recorded extracellularly from teased dorsal root fibers or intracellularly with sharp electrodes from somata of DRG neurons with myelinated (A&bgr; and A&dgr;) or unmyelinated (C) axons. IS increased the rate of ongoing spontaneous activity recorded from dorsal root fibers of CCI neurons and evoked activity in a subpopulation of previously ‘silent’ fibers in CCI rats but not those of unoperated controls. In comparison with DRG somata of control rats, those of CCI become more excitable as evidenced by a lower threshold to depolarizing current and a greater depolarization in response to IS. Inflammatory mediators, by increasing the excitability of DRG neurons, may contribute to paresthesiae, pain and hyperalgesia after peripheral nerve injury.

Spinal Matrix Metalloproteinase-9 Contributes to Physical Dependence on Morphine in Mice

Preventing and reversing opioid dependence continues to be a clinical challenge and underlying mechanisms of opioid actions remain elusive. We report that matrix metalloproteinase-9 (MMP-9) in the spinal cord contributes to development of physical dependence on morphine. Chronic morphine exposure and naloxone-precipitated withdrawal increase activity of spinal MMP-9. Spinal inhibition or targeted mutation of MMP-9 suppresses behavioral signs of morphine withdrawal and the associated neurochemical alterations. The increased MMP-9 activity is mainly distributed in the superficial dorsal horn and colocalized primarily with neurons and small numbers of astrocytes and microglia. Morphine exposure and withdrawal increase phosphorylation of NR1 and NR2B receptors, ERK1/2, calmodulin-dependent kinase II, and cAMP response element binding proteins; and such phosphorylation is suppressed by either spinal inhibition or targeted mutation of MMP-9. Further, spinal administration of exogenous MMP-9 induces morphine withdrawal-like behavioral signs and mechanical allodynia, activates NR1 and NR2 receptors, and downregulates integrin-β1, while a function-neutralizing antibody against integrin-β1 suppresses MMP-9-induced phosphorylation of NR1 and NR2B. Morphine withdrawal-induced MMP-9 activity is also reduced by an nNOS inhibitor. Thus, we hypothesize that spinal MMP-9 may contribute to the development of morphine dependence primarily through neuronal activation and interaction with NR1 and NR2B receptors via integrin-β1 and NO pathways. The other gelatinase, MMP-2, is not involved in morphine dependence. Inhibiting spinal MMP-9 or MMP-2 reduces chronic and/or acute morphine tolerance. This study suggests a novel therapeutic approach for preventing, minimizing, or reversing opioid dependence and tolerance.

Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy

EphrinB–EphB receptor signaling plays diverse roles during development, but recently has been implicated in synaptic plasticity in the matured nervous system and in pain processes. The present study investigated the correlation between expression of ephrinB and EphB receptor proteins and chronic constriction injury (CCI) of the sciatic nerve and dorsal rhizotomy (DR) in dorsal root ganglion (DRG) and spinal cord (SC); and interaction of CCI and DR on expression of these signals. Adult, male Sprague–Dawley rats were employed and thermal sensitivity was determined in the sham operated CCI and DR rats. Western blot and immunobiochemistry analysis and immunofluorescence staining techniques were used to detect the expression and location of the ephrinB–EphB receptor proteins in DRG and SC. The results showed that expression of ephrinB1 and EphB1 receptor proteins was significantly upregulated in DRG and SC in a time‐dependent manner corresponding to the development of thermal hyperalgesia after CCI. The increased expression is predominately located in the medium‐ and small‐sized DRG neurons, the superficial layers of spinal dorsal horn (DH) neurons, and the IB4 positive nociceptive terminals. DR increases ephrinB1 in DRG, not SC and EphB receptor in SC, not DRG. DR suppressed CCI‐induced upregulation of ephrinB1 in SC and EphB1 receptor in DRG and SC. These findings indicate that ephrinB–EphB receptor activation and redistribution in DRG and DH neurons after nerve injury could contribute to neuropathic pain. This study may also provide a new mechanism underlying DR‐induced analgesia in clinic.

EphrinB-EphB receptor signaling contributes to bone cancer pain via Toll-like receptor and proinflammatory cytokines in rat spinal cord.

Summary A new mechanism underlying contributions of ephrinB‐EphB receptor signaling to bone cancer pain: Toll‐like receptor 4‐glial cell pathway. Abstract Treating bone cancer pain poses a major clinical challenge, and the mechanisms underlying bone cancer pain remain elusive. EphrinB‐EphB receptor signaling may contribute to bone cancer pain through N‐methyl‐d‐aspartate receptor neuronal mechanisms. Here, we report that ephrinB‐EphB signaling may also act through a Toll‐like receptor 4 (TLR4)‐glial cell mechanism in the spinal cord. Bone cancer pain was induced by tibia bone cavity tumor cell implantation (TCI) in rats. TCI increased the expression of TLR4 and the EphB1 receptor, the activation of astrocytes and microglial cells, and increased levels of interleukin‐1&bgr; (IL‐1&bgr;) and tumor necrosis factor‐&agr; (TNF‐&agr;). The increased expression of TLR4 and EphB1 were colocalized with each other in astrocytes and microglial cells. Spinal knockdown of TLR4 suppressed TCI‐induced behavioral signs of bone cancer pain. The TCI‐induced activation of astrocytes and microglial cells, as well as the increased levels of IL‐1&bgr; and TNF‐&agr;, were inhibited by intrathecal administration of TLR4‐targeting siRNA2 and the EphB receptor antagonist EphB2‐Fc, respectively. The administration of EphB2‐Fc suppressed the TCI‐induced increase of TLR4 expression but siRNA2 failed to affect TCI‐induced EphB1 expression. Intrathecal administration of an exogenous EphB1 receptor activator, ephrinB2‐Fc, increased the expression of TLR4 and the levels of IL‐1&bgr; and TNF‐&agr;, activated astrocytes and microglial cells, and induced thermal hypersensitivity. These ephrinB2‐Fc‐induced alterations were suppressed by spinal knockdown of TLR4. This study suggests that TLR4 may be a potential target for preventing or reversing bone cancer pain and other similar painful processes mediated by ephrinB‐EphB receptor signaling.

Resveratrol reduces morphine tolerance by inhibiting microglial activation via AMPK signalling

Evidence has accumulated indicating that microglia within the spinal cord play a critical role in morphine tolerance. The present study investigated the effects and possible mechanisms of 5′ adenosine monophosphate‐activated protein kinase (AMPK) activator resveratrol and AICAR to inhibit microglial activation and to limit the decrease in antinociceptive effects of morphine.