ERK-CREB Signal Pathway Contribute To Formation And Maintenance Of Neuropathic Pain In Rats

Varying etiologies-induced peripheral nerve injury may produce chronic pain states characterized by hyperalgesia, allodynia and spontaneous pain. To this date, there have been no effective treatments that can release neuropathc pain, and its mechanisms are also unclear. Recently, synaptic plasticity of spinal cord neurons induced by long-lasting nociceptive stimulates (also called central sensitization) is under intensive investigation. Several studies have suggested that central sensitization related to pain and hippocampus long term potentiation (LTP) associated with learning and memory may share certain similar mechanisms. For example, activation of NMDA receptor and subsequent associated intracellular signal transduction cascades are involved in induction, development and maintainment of synaptic plasticity in the spinal cord and hippocampus. Like the consolidation of early-phase LTP into late-LTP in hippocampus, activity-dependent gene expression or transcription, which can increase the expression of pain-related receptor and signal protein, plays an important role during converting from acute nociceptive injury to chronic pain states. The transcription factor cAMP response element binding protein (CREB), which could be phosphorylated by multiple intracellular kinases in response to a vast of physiological and pathological stimuli, is critical for activity-dependent gene expression. The genetic studies have showed that CREB contributes to hippocampus late– LTP and memory consolidation and CREB activation may serve as a molecular switch to transform short-lasting into long-lasting synaptic plasticity in the hippocampus. Similarly, CREB has been suggested to contribute to central sensitization associated with persistent pain states. It has been proposed that NMDA activation-induced Ca++ influx can trigger an early phase of CREB phosphorylation and a persistent phase of CREB phosphorylation mediates by a delayed ERK/MAPK signal cascade, which is important to the development and maintainment of chronic pain.Extracellular signal-regulated kinase (ERK), one member of the mitogen-activated protein kinases (MAPK) family, transduces a broad range of extracellular stimuli into diverse intracellular responses by producing changes in the level of gene expression or transcription. Activated ERK translocates from the cytosol into the nucleus and in turn phosphorylates CREB at serine residue 133. Phosphorylation of CREB binding to cAMP response element (CRE) of target gene regulates gene expression and mediates roles of ERK. A number of studies have shown that ERK-mediated CREB phosphorylation is required for the induction of stable, late-phase LTP and long-term memory. It has been reported that ERK may be involved in modulation of nociceptive information and central sensitization produced by intense noxious stimuli and/or peripheral tissue inflammation. However, few studies have focused on the roles of ERK and the relationship between ERK and CREB in neuropathc pain produced by nerve injury, such as chronic constriction injury (CCI) of the sciatic nerve.In the present study, we used CCI model to investigate (1) whether activation and translocation of ERK was involved in induction and maintenance of chronic neuropathic pain, and (2) effect of activation and translocation of ERK on expression of CREB in chronic neuropathic pain.We observe and measure the behavioral changes by von Frey filaments and thermal stimulus in CCI rats, we also observe the expression of ERK1/2, pERK1/2, pCREB and c-fos in spinal cord dorsal horn by immunohistochemistry and western blotting methods. Meanwhile, observe the effect of Pretreatment or single intrathercal injection ERK signal pathway inhibitor U0126 or ERK1/2 antisense ODN on the behavioral results and expression of ERK1/2, pERK1/2, pCREB and c-fos in spinal cord dorsal horn.Findings:1. Intrathecal injection of U0126 or ERK antisense ODN attenuated or reversed CCI-induced mechanical and thermal hyperalgesia. Intrathecal administration of U0126 and ERK antisense-ODN did not affect the mechanical and thermal threshold of PWL and motor functions in the rats2. CCI significantly increased the expression of p-ERK-IR neurons in the spinal dorsal horn ispilateral to injury. The p-ERK-positive neurons distributed mainly in laminaeⅠ,Ⅱ. Few positive neurons distributed in other laminae and the contralateral spinal cord. Intrathecal injection of U0126 and ERK antisense ODN inhibited the increase of ERK expression in spinal dorsal horn. Intrathecal injection of U0126 or ERK antisense ODN markedly inhibited the increase of both cytosolic and nuclear pERK expression. The changes of pERK expression were correlated with the observed behavior hyperalgesia.3. The Immunohistochemical and western blotting results revealed that CCI can significantly increased the expression of pCREB and pCREB positive neurons distributed all laminae of bilateral spinal cord. The expression in ispilateral and contralateral spinal cord showed no significant different. Intrathecal injection of U0126 or ERK antisense ODN markedly inhibited or reversed pCREB expression in both sides of the spinal cord.4. CCI increased significantly the expression of c-Fos in laminaeⅠ-Ⅱof the ipsilateral spinal dorsal horn. Unlike the expression of pCREB, few c-Fos positive neurons expressed in the contralateral spinal cord. Intrathecal administration of U0126 or ERK antisense ODN also significantly reduced or reversed the expression of c-Fos positive neurons.Central sensitization, an activity-dependent functional plasticity, is one of main cause for behavior hyperalgesia under pathological conditions. Postsynaptic membrane receptors and ion channels activation, intracellular kinase cascades and intraneclear gene expression contribute to the induction, development and maintainment of central sensitization. Intracellular kinase cascades, as the linkage bridge, transduce noxious stimuli into diverse intracellular responses, including changes in level of gene expression or transcription. A large number of studies have shown that several protein kinases implicated in central sensitization, for example, protein kinases A (PKA), protein kinases C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). Recently, several studies have reported that ERKs, a mitogen-activated protein kinase, contributes to pain hypersensitivity and central sensitization. Acute noxious stimuli, such as formalin or capsaicin, induced ERK phosphorylation in spinal dorsal horn neurons and MEK inhibitor PD 98059 or U0126 reduced acute pain behavior after subcutaneous injection of formalin or capsaicin. The present studies indicate that activation of ERK in rats spinal cord contributed to CCI-induced hyperalgesia. This is in agreement with the previous reports in other pain model, We demonstrated, for the first time, the time course of pERK and relationship of pERK and pCREB expression in CCI model. CCI could induce a long-lasting ERK phosphorylation and pERK translocation into nuclear in spinal dorsal horn neurons. The time course of pERK correlated significantly with behavior hyperalgesia and intrathecal injection of U0126 or ERK antisense ODN attenuated significantly CCI-induced mechanical and thermal hyperalgesia.Nerve fibers produce abnormal ectopic excitability at or near the site of nerve ligation after CCI. The local persistent abnormal excitability of sensory nerve can spread to distant parts of the peripheral including the peripheral nerve bodies in the dorsal root ganglion (DRG) and central nervous system. Repeated or prolonged noxious stimulation and the persistent abnormal input following nerve injury increase the release of nociceptive neurotransmitter, such as glutamate, substance P, calcitonin gene-related peptide (CGRP) in the central terminals of primary sensory afferents and then activate NMDA and NK receptor in spinal dorsal horn neurons. Calcium influx through NMDA receptor triggers an increase in the levels of Ras-GTP and in turn leads to the activation of raf/MEK/ERK cascades.ERK mediates central sensitization by phosphorylating effector protein. Two potential effectors of ERK related to central sensitization are the potassium channel Kv4.2 and the transcription factor CREB. The recent evidence showed that ERK integrated PKA and PKC to modulate the K+ channel Kv4.2 and A-type K+ currents (IA) in superficial dorsal horn neurons. Kv4.2 and IA are critical determinants of neuronal excitability in the central sensitization. It is possible that modulation of potassium channel may involve in the short-lasting or acute effect of pERK. Our data shew that the CCI-induced hyperalgesia could be reversed 30 min after intrathecal administration of U0126 on postoperative 5 days.Importantly, pERK could translocate from cytoplasm into nuclear and in turn phosphorylate transcriptional factors CREB on serine 133. In the current study, CCI increased significantly the expression of pCREB in the rat spinal cord. The time course of pCREB expression correlates with activation of ERK and behavior hyperalgesia. Intrathecal injection of U0126 or ERK antisense ODN markedly inhibited the increase of pCREB expression. It suggestes that activation of ERK may contribute to the increased pCREB expression in the spinal cord of CCI rats, and function of pERK is partly accomplished via CREB-dependent gene expression. Phosphorylation of CREB on serine 133 recruits the CREB binding protein, CBP, to the complex and promotes transcription of downstream genes. Many"pain genes", which may contribute to central sensitization, are activated by CREB, including the immediate early gene c-fos, BDNF, CGRP, the alpha subunit of CaMKII and neurokinin 1 receptor. A considerable amount of evidences have indicated that CREB-dependent gene expression was required for long-term changes in synaptic plasticity induced by various nociceptive stimuli.In summary, activations of ERK, CREB and c-Fos show similar time-courses in spinal cord dorsal horn of CCI rats which corrected to behavior hyperalgesia. Intrathecal injections of U0126 or ERK antisense oligodeoxynucleotides can inhibit the expression of pERK, pCREB and c-Fos, significantly attenuate or reverse CCI-induced mechanical and thermal hyperalgesia. The present studies suggest that activation of ERK/MAPK may contribute to CCI-induced pain and hyperalgesia, and function of pERK may partly be accomplished via CREB-dependent gene expression.