The Role Of Spinal EprhinB-EphB Signaling In Chronic Pain And Morphine Tolerance And Withdrawal

Background and ObjectivesChronic pain is difficult to cure and easily leads to irritability, depression and othernegative emotions so it greatly limits the patient’s daily activities and labor ability.Patients with chronic pain are suffering long-term heavy burden in both body and spirit.The quality of life is severely compromised. According to the World Health Organizationreports that12to15hundred million people in the world are suffering from chronic pain,and with the arrival of the worldwide "aging of population", this number will furtherincrease.Although a wide range of treatment means of chronic pain are ready to be chosen,such as physical therapies (laser, infrared, electromagnetic, etc.), analgesic drugs (opioids,anti-inflammatory drugs, sedative drugs) and surgery etc., but it is still hard to havechronic pain well controled. An obvious reason for this situation is that mechanismsunderlying chronic pain remain eclusive. Thus, the pain treatment is blinded, empiricaland targetless. Therefore, to clarify the mechanism of chronic pain and to develop better analgesic technique and drugs are the two primary issues for basic researchers, andclinicians.This study focuses on both underlying mechanisms and drug targets discovery.Firstly, we observed that blocking EphB receptors results in ideal analgesic effect on theanimals with neuropathic pain produced by CCI and with cancer pain produced by tumorcell transplantation (TCI). These two models are well mimicking injury in clinic. secondly,we have found that blocking EphB receptor signaling inhibited hyperexcitability of DRGneurons and spinal dorsal horn neurons, as well as decrease of NMDAR phosphorylation;thirdly, we found that EphB receptor signaling pathways has similar role in morphinetolerance and dependence, suggesting that blocking EphB receptor signaling is helpful toovercome the side effects of morphine, and to expand its clinical application; Finally, wehave identified that EphB receptor subtype EphB1may play important roles in theseprocesses by selectively knockout the specific genes. In this study, so far, we have found aspecific pharmaceutical target for further investagation and laid a solid foundation forfurther research on drug development.Part: ephrinB-EphB receptor signaling in chronic pain and its MechanismObjectiveTo demonstrate the effect of ephrinB-EphB receptor signaling on chronic pain andits mechanismMethodWe selected classic chronic constriction injury (CCI) animal model and bonecancer pain animal model in this article.EphB receptor blockers (EphB-Fc) was used to block the spinal dorsal horn EphBreceptor signaling, and subsequently we detected thermal hyperalgesia (noxioushyperalgesia) and mechanical allodynia (non-noxious hyperalgesia) by radiant heatmethod and Von Frey method sepereatly; Finally, we used statistical analysis to analyzethe data of pain behavior induced by CCI. According to different administration-style ofEphB-Fc, animals were divided into several groups:1) in development period (1-3daysafter operation) or in maintainance period (3days after operation) of CCI-inducedhyperalgesia to observe whether there is a different of effect of inhibition of EphBreceptor signaling between the two periods;2) single dose administration group was aimed to observe the duration of drug action and three consecutive administration group aimedto observed superimposed effect or tolerance effect.Secondly, we selected breast cancer cells Walker256bone cancer pain modelwhich was closer to clinical practice. Using the same test methods mentioned above, wetested the impact of three consecutive administration of EphB-Fc on thermal hyperalgesiaand mechanical allodynia induced by bone cancer pain;Thirdly, we used the western blot method to detect ephrinB and EphB expressiontrend in spinal cord and dorsal root ganglia in CCI model and bone cancer pain model, andthe immunofluorescence method was used to further clarify distribution and cellularlocalization of ephrinB and EphB in the DRG and spinal dorsal horn.Then, we used in vitro intact DRG neuron patch clamp recording technology toresearch the impact of EphB signaling in CCI injury induced DRG neurons’ hyper-excitement, the record indicators of the excitability were action potential threshold value,quantity of evoked action potentials and the number of neurons of occurrence ofspontaneous action; single unit recording technique in wide dynamic range (WDR)neurons of spinal cord was used to research EphB signal the CCI injury induced spinalneuron hyper-excitement. When we applied brush (light), holder (medium) and clamp(heavy) three different intensity (quantified by force per square millimeter) stimulation tofoot, WDR neurons spike more in CCI group than in sham group; induction of LTPbetween C fibers and spinal cord dorsal horn neurons technique was used to researchimpact of EphB receptor signaling in LTP induction. This experiment reflected the role ofEphB receptor signaling in synaptic plasticity.Finally, behavioral test and western blot were used to investigate whether EphBsignaling regulate NMDAR phosphorylation.ResultsPreventive administration (in development period of hyperalgesia) andtreatmentive (in maintenance period of hyperalgesia) administration of EphB receptorsignaling blockers (EphB-Fc) could block the nerve injury (CCI)-induced thermalhyperalgesia and mechanical allodynia; single dose postponed hyperalgesia formation fortwo days, while preventive three consecutive administration completely inhibited theformation of hyperalgesia; therapeutic administration of single-dose inhibited thehyperalgesia for24hrs; and for three consecutive administration, the efficacy disappeared in24hour after the end of administration, without superimposing and tolerance effects;Since in bone cancer pain hyperalgesia was settled at the7th postoperative day,preventive administration time of EphB-Fc was adjusted to postoperative3,4,5day andthe time for therapeutic administration is postoperative7,8,9day. Both preventive andtherapeutic administration of EphB-Fc effectively suppressed thermal hyperalgesia andmechanical allodynia induced by bone cancer.Both single-dose injection and three consecutive injection of EphB receptorsignaling agonists (ephrinB-Fc) in naive rats caused thermal hyperalgesia, and the WesternBlot results showed there was upregulation of phosphorylated EphB receptor. Theseresults indicated that ephrinB-Fc activated EphB receptor signaling and EphB receptorsignaling was involved in the occurrence of chronic pain.The above results show that EphB receptor signaling involved in the occurrenceand development process of neuropathic pain and bone cancer pain. Inhibition of EphBreceptor signaling can relieve chronic pain. Next, we will research the underlyingmechanisms of EphB receptor signaling in chronic pain.In CCI and bone cancer pain models, Western Blot was used to show ephrinB andEphB protein expression in dorsal root ganglion (DRG) and spinal cord during thedevelopment process of chronic pain; and ephrinB and EphB receptor expression wereconsistent with the timecourse of hyperalgesia, which suggested that there was acorrelation between the protein expression and hyperalgesia; the immunofluorescencestain also showed that ephrinB and EphB expressed in DRG and spinal cord dorsal horn;double immunofluorescent labeling showed that ephrinB and EphB had similar cellurlocation, which mainly expressed in DRG neurons, including large, medium and smallsize neurons and co-localizated with CGRP-posivitive neurons,while in the spinal cord,ephrinB and EphB expressed in neurons and astrocytes.Injury caused by the CCI induced hyper-excitability in DRG neurons which wasshowed as declining in action potential threshold, increasing induced action potentialfrequency generation and increasing spontaneous discharge activities; and CCI injury alsoincreased the reflection of wide dynamic range (WDR) in spinal cord to the peripheralstimulation. EphB-Fc blocked the CCI-induced DRG and spinal dorsal horn WDRneurons’ excitability. LTP between C fibers and spinal cord dorsal horn neurons reflectssynaptic plasticity in the C fibers and spinal dorsal horn synapses, EphB-Fc couldpreventive block the LTP between C fibers and spinal dorsal horn neurons, which suggested that EphB1-Fc was involved in the regulation of chronic pain through regulatingsynaptic sensitization happened in the spinal cord.The intrathecal injection EphB receptor signaling agonists (ephrinB-Fc) in Naiveanimals produced thermal hyperalgesia and induced phosphorylation of EphB1receptorand NMDAR associated molecules in spinal dorsal horn, while the thermal hyperalgesiaand increased phosphorylation degree of EphB1and NMDAR-related signaling moleculeswere blocked by specific NDMAR inhibitor-MK-801. Similarly, in the TCI model, EphB-Fc inhibited the activation TCI induced phosphorylation of NMDAR signaling pathway(NR1, NR2B, src, ERK, CaMK and CREB). Above results suggest that EphB receptorsignaling pathways involved in the regulation of the NMDAR.The above experiments show how the EphB receptor signal pathway is involved inchronic pain, that is, EphB singnaling regulate the DRG and spinal cord dorsal hornneuronal excitability and NMDAR-related synaptic plasticity processes betweenperipheral nerve and spinal cord neurons.Part: ephrinB-EphB receptor signaling in morphine tolerance and withdrawalObjectiveThe opioid drugs are most effective medicine in current clinical treatment ofintractable pain, but it’s shortcomings of tolerance and dependence limits its application inclinic. Morphine tolerance and dependence mechanism studies have shown that chronicpain has a very similar mechanism with chronic pain, with glutamate receptor-mediatedcentral sensitization; therefore, we explore ephrinB-EphB role in morphine tolerance andmorphine withdrawal process after investigating its role in chronic pain.MethodTo study the EphB receptor signaling in morphine tolerance, we firstly, in acuteand chronic morphine tolerance model, preventive (in acute model given morphine for thefirst30minutes; in chronic models each given morphine for the first30minutes)administrate EphB receptor inhibitors (EphB-Fc) to observe the behavior of the modelswith indicator of maximum percentage of analgesic effect (MPE%); and Western Blottechnique was used to observed EphB activation(pEphB) of receptor signaling inmorphine tolerance; at last, we test the analgesic effect of morphine in the bone cancerpain model with the combinated administration of morphine and EphB-Fc. In study of EphB receptor signaling in morphine withdrawal, firstly we test theimpact of preventive administration of EphB1-Fc in naloxone-induced morphinewithdrawal animal model with separate indicators of backward walking, chewing, diarrhea,jumps, paw tremor, ptosis, tremor, wet dog shaking and weight loss ect. and the compositescore derived by separate indicators; in addition, we use western blot technique to detectEphB1receptor expression and activation in the morphine withdrawal process. The resultssuggest that EphB receptor signaling participation in the above process; Shortly thereafter,we quantitatively observed intrathecal EphB-Fc spinal cord c-fos expression and CGRP byimmunofluorescence; finally, Western Blot techniques we use to detect change ofNMDAR and its related molecules, such as pNR2B, pERK and pCREB before and afteradministration of EphB-Fc.ResultsPreventive single-dose of EphB-Fc can increase the maximum analgesic effect ofmorphine percentage (%MPE) in acute or chronic morphine tolerance model; and theactivated EphB receptor signaling in acute or chronic morphine tolerance model wereobserved, indicating that EphB signaling is indeed involved in the formation of morphinetolerance; EphB-Fc also blocked morphine tolerance formation in bone cancer pain model;Preventive administration of EphB-Fc can alleviate morphine withdrawalbehavioral performance, and EphB-Fc down-regulated the expression of c-fos and CGRPwhich were upregulated in morphine application process; and morphine withdrawal causesa large number of CGRP release (downregulation)in the spinal cord dorsal horn, whilepreventive administration of EphB-Fc alleviate CGRP downregulation. The preventiveadministration of EphB-Fc aslo reduced elevated NMDAR phosphorylation levelsincreased in morphine and withdrawal process.From above experiments, we conclude that:Conclusions1. EphB receptor signaling is involved in chronic pain; inhibiting EphB signalingcould reduce chronic pain.2. EphB receptor signaling is involved in morphine tolerance and withdrawal;inhibiting EphB signaling couled release morphrine tolerance and withdrawal. Ourexperiment results help to expand the morphine usage in clinic.