The Function Of NR2B Receptor In Peripheral Hyperalgesia On Chronic Post-Ischemia Pain (CPIP) Model

Introduction:Complex regional pain syndrome(CRPS)is a chronic,refractory clinical condition featured by limb hyperalgesia,which gravely undermines patients’life quality.NR2B plays an important role in neuropathis pain in CCI and other models.Previous studies have shown that keratinocyte activation plays an important role in peripheral transmission of pain which can be alleviated by the expression of N-methyl-D-aspartic acid(NMDA)receptor on keratinocytes.Based on the research done in our laboratory before that NMDA receptors in keratinocytes are associated with pain in CRPS.Upregulation of NMDA receptor expression in keratinocytes occurs in CRPS both acute and chronic phases.In the acute phase of CRPS,NMDA receptors in keratinocytes regulate skin pro-inflammatory cytokine release and pain conduction,while in the CRPS chronic phase,keratinocyte NMDA receptor regulates activation of microglia and astrocytes in spinal dorsal horn,and release of pro-inflammatory cytokines in spinal cord.This study is aimed to further explore the role of NMDA receptor subunit NR2B in CRPS hyperalgesia,with special attention to its expression in Dorsal Root Ganglion(DRG)-bridge of skin and spinal cord pain transmission in CRPS acute and chronic phase,and the effect on related signaling pathways and pro-inflammatory factors.Methods:①Grouping and interventions:Rat chronic post-ischemia pain(CPIP)model,a well-recognized animal model of CRPS,was applied in this study.Subcutaneous injection of NMDA receptor agonist NMDA and NR2B antagonist ifenprodil was adopted to study the function of NMDA receptor in keratinocytes.Firstly,male Sprague-Dawley(SD)rats were divided into acute and chronic phase group.The acute group was given drugs for consecutive 3 days before modeling,while drug administration was started from the 7th day after modeling to the 14th day in chronic group.Both groups were subdivided into Naive group,Sham group,and CPIP group.In CPIP group,an O-ring(inner diameter=0.65 cm)was placed tightly on the ankle of the right limb of rats and removed 3 hours later.Contrarily,a cut O-ring was loosely placed in Sham group and no treatment in Naive group.The CPIP group continued to be subdivided into 3 groups,CPIP+NMDA group,CPIP+IFEN(ifenprodil)group,CPIP+NS(normal saline)group,and administered NMD A(1 mM),ifenprodil(10μg)and normal saline 100μl daily,respectively.No treatment in non-intervention groups after modeling.② Behavior and physiology test:the electronic von-Frey was used to test mechanical sensitivity and heat-plate was used to test thermal sensitivity.The tests were performed at the 2nd,1st,and 0th days before modeling,and the 6th,12th,18th and 24th hour after modeling in acute groups and on the 2nd,1st day before modeling and the 2nd,6th,10th,14th day after modeling in chronic groups.(3)Tissue extraction:The right hind paw skin and L2-L4 spinal cord was harvested on the 1st day after modeling in acute groups,the 14th day in chronic groups,and the 1st,6th and 10th day after modeling in non-intervention groups.④Western Blot:Expression of pNR2B,NR2B,pNF-KB,NF-κB,pErk,Erk,IL-1β,TNF-a in skin and spinal cord was determined.⑤Immunofluorescences staining:Co-expression of keratinocyte marker pan-keratin with NR2B,pNR2B,IL-1β,TNF-a in skin tissue,and co-expression of the neuronal marker NeuN with NR2B,pNR2B,pNF-KB,IL-1β,TNF-a in spinal cord sample were determined.Results:①Behavior and physiological tests:The comparison of von-Frey and heat-plate tests in both acute and chronic phase was CPIP+NMDA<CPIP+NS<CPIP+IFEN<Sham.②Skin Immunofluorescences staining and Western Blot:In both acute and chronic phase,the comparison of NMDA receptor expression on keratinocytes was CPIP+NMDA>CPIP+NS>Sham.The comparison of TNF-a and IL-1β release in acute phase was CPIP+NMDA>CPIP+NS>CPIP+ IFEN/Sham.This trend failed to persist in chronic phase.③ DRG Immunofluorescences staining and Western Blot:The comparison of NR2B and pNR2B receptors expression:CPIP+NMDA>CPIP+NS>CPIP+ IFEN/Sham.The comparison of pNF-κB,TNF-a and IL-1β expression in acute and chronic phase:CPIP+NMDA>CPIP+NS>CPIP+ IFEN/Sham.The expression levels of pNF-KB and pErk in DRG gradually increased on the 1st,6th and 10th days.Conclusions:①NR2B is involved in the pathophysiological process of CRPS.The expression level and activation of NR2B receptor in skin keratinocytes are closely related to CRPS pain.Subcutaneous injection of ifenprodil before CRPS and continuous injection of ifenprodil after CRPS are both effective in reducing CRPS pain,②Acute phase of CRPS:Activation of NR2B in the skin increased release of inflammatory cytokines,leading to peripheral sensitization.The transition of pain into DRG resulted in activation of NR2B on DRG neurons,which gradually activated pNF-KB,pERK signaling pathway and inflammatory factors release.DRG pain into the spinal cord can not yet cause glial activation and inflammatory factors release.③Chronic phase of CRPS:The NR2B that has been expressed by the skin keratinocytes persistently exist and has not yet been transformed,but the inflammatory factors of the skin are relatively reduced.The NR2B on DRG was persistently activated,leading to the activation of pNF-KB and pERK signaling pathways and the release of a large number of inflammatory factors.DRG pain is further transmitted to the spinal cord,causing glial activation and release of inflammatory factors,leading to central sensitization.