Jun-Li Cao

Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury-induced neuropathic pain in rats.

AbstractAim:To investigate whether activation and translocation of extracellular signal-regulated kinase (ERK) is involved in the induction and maintenance of neuropathic pain, and effects of activation and translocation of ERK on expression of pCREB and Fos in the chronic neuropathic pain.Methods:Lumbar intrathecal catheters were chronically implanted in male Sprague-Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciaticas trifurcation at approximately 1.0 mm intervals with 4-0 silk sutures. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or phosphorothioate-modified antisense oligonucleotides (ODN) were intrathecally administered every 12 h, 1 d pre-chronic constriction injury (CCI) and 3 d post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The expression of pERK, pCREB, and Fos were assessed by both Western blotting and immunohistochemical analysis.Results:Intrathecal injection of U0126 or ERK antisense ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of p-ERK-IR neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK expression showed that the levels of both cytosol and nuclear pERK, but not total ERK, were increased at all points after CCI and reached a peak level on postoperative d 5. CCI also significantly increased the expression of pCREB and Fos. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord and Fos was expressed in laminae I and II of the ipsilateral spinal dorsal horn. Intrathecal injection of U0126 or ERK antisense ODN markedly suppressed the increase of CCI-induced pERK, pCREB and c-Fos expression in the spinal cord.Conclusion:The activation of ERK pathways contributes to neuropathic pain in CCI rats, and the function of pERK may partly be accomplished via the cAMP response element binding protein (CREB)-dependent gene expression.

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.

Activation of the spinal ERK signaling pathway contributes naloxone-precipitated withdrawal in morphine-dependent rats.

&NA; Extracellular signal‐regulated kinase (ERK), a mitogen‐activated protein kinases (MAPK), transduces a broad range of extracellular stimuli into diverse intracellular responses. Recent studies have showed that ERK activation in the supraspinal level involved in the development of drug dependence, especially in psychological dependence. In this study, we reported that the spinal ERK signaling pathway was activated by chronic morphine injection. There was a further increase in ERK activation after naloxone‐precipitated withdrawal. Furthermore, attenuation of the spinal ERK phosphorylation by intrathecal a MAPK kinase (MEK) inhibitor U0126 or knockdown of the spinal ERK by antisense oligonucleotides not only decreased the scores of morphine withdrawal, but also attenuated withdrawal‐induced allodynia, which were accompanied by decreased ERK phosphorylation in the spinal cord. The spinal ERK inhibition or knockdown also reduced morphine withdrawal‐induced phosphorylation of cAMP response element binding protein (CREB), which is one of the important downstream substrates of ERK pathway, and Fos expression. The involvement of the spinal ERK in morphine withdrawal was supported by our finding that intrathecal N‐methyl‐d‐aspartate receptor antagonist MK‐801 or protein kinase C inhibitor chelerythrine chloride suppressed withdrawal‐induced ERK activation in the spinal cord and attenuated morphine withdrawal symptoms. These findings suggest activation of the spinal ERK signaling pathway contributes naloxone‐precipitated withdrawal in morphine‐dependent rats.

Epigenetic Modification of Spinal miR-219 Expression Regulates Chronic Inflammation Pain by Targeting CaMKIIγ

Emerging evidence has shown that miRNA-mediated gene expression modulation contributes to chronic pain, but its functional regulatory mechanism remains unknown. Here, we found that complete Freunds adjuvant (CFA)-induced chronic inflammation pain significantly reduced miRNA-219 (miR-219) expression in mice spinal neurons. Furthermore, the expression of spinal CaMKIIγ, an experimentally validated target of miR-219, was increased in CFA mice. Overexpression of spinal miR-219 prevented and reversed thermal hyperalgesia and mechanical allodynia and spinal neuronal sensitization induced by CFA. Concurrently, increased expression of spinal CaMKIIγ was reversed by miR-219 overexpression. Downregulation of spinal miR-219 in naive mice induced pain-responsive behaviors and increased p-NMDAR1 expression, which could be inhibited by knockdown of CaMKIIγ. Bisulfite sequencing showed that CFA induced the hypermethylation of CpG islands in the miR-219 promoter. Treatment with demethylation agent 5′-aza-2′-deoxycytidine markedly attenuated pain behavior and spinal neuronal sensitization, which was accompanied with the increase of spinal miR-219 and decrease of CaMKIIγ expression. Together, we conclude that methylation-mediated epigenetic modification of spinal miR-219 expression regulates chronic inflammatory pain by targeting CaMKIIγ.

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.

Molecular Mechanisms Underlying the Analgesic Property of Intrathecal Dexmedetomidine and Its Neurotoxicity Evaluation: An In Vivo and In Vitro Experimental Study

Background Dexmedetomidine (DEX) has been used under perioperative settings as an adjuvant to enhance the analgesic property of local anesthetics by some anesthesiologists. However, the analgesic mechanisms and neurotoxicity of DEX were poorly understood. This study examined the effect of DEX alone on inflammatory pain, and it also examined the underlying molecular mechanisms of DEX in the spinal cord. Furthermore, in vivo and in vitro experiments were performed to investigate the neurotoxicity of DEX on the spinal cord and cortical neurons. Methods This study used adult, male Kunming mice. In the acute inflammatory model, the left hind-paws of mice were intradermally injected with pH 5.0 PBS while chronic constrictive injury (CCI) of the sciatic nerve was used to duplicate the neuropathic pain condition. Thermal paw withdrawal latency and mechanical paw withdrawal threshold were tested with a radiant heat test and the Von Frey method, respectively. Locomotor activity and motor coordination were evaluated using the inverted mesh test. Western blotting examined spinal ERK1/2, p-ERK1/2, caspase-3 and β-actin expressions, while spinal c-Fos protein expression was realized with immunohistochemical staining. Hematoxylin eosin (HE) staining was used to examine the pathological impacts of intrathecal DEX on the spinal cord. DAPI (4′,6-diamidino-2-phenylindole) staining was used to observe cell death under an immunofluorescence microscope. Results Intra-plantar pH 5.0 PBS-induced acute pain required spinal ERK1/2 activation. Inhibition of spinal ERK1/2 signaling by intrathecal injection of DEX displayed a robust analgesia, via a α2-receptor dependent manner. The analgesic properties of DEX were validated in CCI mice. In vivo studies showed that intrathecal DEX has no significant pathological impacts on the spinal cord, and in vitro experiments indicated that DEX has potential protective effects of lidocaine-induced neural cell death. Conclusion Intrathecal injection of DEX alone or as an adjuvant might be potential for pain relief.

EphrinBs/EphBs Signaling Is Involved in Modulation of Spinal Nociceptive Processing through a Mitogen-activated Protein Kinases-dependent Mechanism

Background:Our previous studies have demonstrated that EphBs receptors and ephrinBs ligands were involved in modulation of spinal nociceptive information. However, the downstream mechanisms that control this process are not well understood. The aim of this study was to further investigate whether mitogen-activated protein kinases (MAPKs), as the downstream effectors, participate in modulation of spinal nociceptive information related to ephrinBs/EphBs. Methods:Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and von Frey filaments test. Immunofluorescence staining was used to detect the expression of p-MAPKs and of p-MAPKs/neuronal nuclei, or p-MAPKs/glial fibrillary acidic protein double label. C-Fos expression was determined by immunohistochemistry. The expression of p-MAPKs was also determined by Western blot assay. Results:Intrathecal injection of ephrinB1-Fc produced a dose- and time-dependent thermal and mechanical hyperalgesia, accompanied by the increase of spinal p-MAPKs and c-Fos expression. Immunofluorescence staining revealed that p-MAPKs colocalized with the neuronal marker (neuronal nuclei) and the astrocyte marker (glial fibrillary acidic protein). Inhibition of MAPKs prevented and reversed pain behaviors and the increase of spinal c-Fos expression induced by intrathecal injection of ephrinB1-Fc. Inhibition of EphBs receptors by intrathecal injection of EphB1-Fc reduced formalin-induced inflammation and chronic constrictive injury-induced neuropathic pain behaviors accompanied by decreased expression of spinal p-MAPKs and c-Fos protein. Furthermore, pretreatment with MK-801, an N-methyl-d-aspartate receptor antagonist, prevented behavioral hyperalgesia and activation of spinal MAPKs induced by intrathecal injection of ephrinB1-Fc. Conclusions:These results demonstrated that activation of MAPKs contributed to modulation of spinal nociceptive information related to ephrinBs/EphBs.

PI3K Contributed to Modulation of Spinal Nociceptive Information Related to ephrinBs/EphBs

There is accumulating evidence to implicate the importance of EphBs receptors and ephrinBs ligands were involved in modulation of spinal nociceptive information. However, the downstream mechanisms that control this process are not well understood. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K), as the downstream effectors, participates in modulation of spinal nociceptive information related to ephrinBs/EphBs. Intrathecal injection of ephrinB1-Fc produced a dose- and time-dependent thermal and mechanical hyperalgesia, accompanied by the increase of spinal PI3K-p110γ, phosphorylation of AKT (p-AKT) and c-Fos expression. Pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented activation of spinal AKT induced by ephrinB1-Fc. Inhibition of spinal PI3K signaling dose-dependently prevented and reversed pain behaviors and spinal c-Fos protein expression induced by intrathecal injection of ephrinB1-Fc. Inhibition of EphBs receptors by intrathecal injection of EphB1-Fc reduced formalin-induced inflammation and chronic constrictive injury-induced neuropathic pain behaviors accompanied by decreased expression of spinal PI3K,p-AKT and c-Fos protein. Furthermore, pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented ephrinB1-Fc-induced ERK activation in spinal. These data demonstrated that PI3K and PI3K crosstalk to ERK signaling contributed to modulation of spinal nociceptive information related to ephrinBs/EphBs.

Phosphatidylinositol 3-kinase mediates pain behaviors induced by activation of peripheral ephrinBs/EphBs signaling in mice

EphBs receptors and their ephrinBs ligands are present in the adult brain and peripheral tissue and play a critical role in modulating multiple aspects of physiology and pathophysiology. Our recent evidence has shown that ephrinBs acted as a sensitizer to participate in peripheral sensitization and hyperalgesia induced by activation of peripheral ephrinBs/EphBs signaling. In the present study, we explored the role of phosphatidylinositol 3-kinase (PI3K) in ephrinB1-Fc-induced pain behaviors. Intraplantar injection of ephrinB1-Fc produced a time- and dose-dependent increase of PI3K-p110gamma expression and of phosphorylation of AKT in skin of injection site. Pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented activation of peripheral AKT by ephrinB1-Fc. The activated AKT expressed in peripheral nerve terminals and DRG peptide-containing and small non-peptide-containing neurons. Inhibition of peripheral PI3K signaling dose-dependently prevented and reversed pain behaviors and spinal Fos protein expression induced by intraplantar injection of ephrinB1-Fc. Furthermore, pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented ephrinB1-Fc-induced ERK activation in a dose-dependent manner. These data demonstrated that PI3K and PI3K crosstalk to ERK signaling mediated pain behaviors induced by activation of peripheral ephrinBs/EphBs signaling in mice.

Activation of peripheral ephrinBs/EphBs signaling induces hyperalgesia through a MAPKs-mediated mechanism in mice.

Abstract EphBs receptors and ephrinBs ligands are present in the adult brain and peripheral tissue and play a critical role in modulating multiple aspects of physiology and pathophysiology. Ours and other studies have demonstrated that spinal ephrinBs/EphBs signaling was involved in the modulation of nociceptive information and central sensitization. However, the role of ephrinBs/EphBs signaling in peripheral sensitization is poorly understood. This study shows that intraplantar (i.pl.) injection of ephrinB1‐Fc produces a dose‐ and time‐dependent thermal and mechanical hyperalgesia and the increase of spinal Fos protein expression in mice, which can be partially prevented by pre‐treatment with EphB1‐Fc. EphrinB1‐Fc‐induced hyperalgesia is accompanied with the NMDA receptor‐mediated increase of expression in peripheral and spinal phosphorylated mitogen‐activated protein kinases (phospho‐MAPKs) including p‐p38, pERK and pJNK, and also is prevented or reversed by the inhibition of peripheral and spinal MAPKs. Furthermore, in formalin inflammation pain model, pre‐inhibition of EphBs receptors by the injection of EphB1‐Fc reduces pain behavior, which is accompanied by the decreased expression of peripheral p‐p38, pERK and pJNK. These data provide evidence that ephrinBs may act as a prominent contributor to peripheral sensitization, and demonstrate that activation of peripheral ephrinBs/EphBs system induces hyperalgesia through a MAPKs‐mediated mechanism.