Mechanism Of Neuropathic Pain By Spinal Paired Immunoglobulin-Like Receptor B

Objective: In this study,the expression and localization of spinal PirB were observed in a mouse model of spared nerve injury induced neuropathic pain,the potential ligands of spinal PirB and other interacting moleculars of downstream were explored,and the effect of spinal PirB on plasticity of spinal excitatory synapses was investigated,to figured out the role and possible mechanism of spinal Pirb in the development of neuropathic pain.Methods:The SNI neuropathic pain model of mice was established and the behavioral changes were observed and evaluated.The expression of the m RNA and protein expression of PirB were detected by Real-time PCR and Western blot.PirB of the spinal cord section was shown by immunohistochemical and immunofluorescence staining.PirB immunofluorescence co-stained with cell membrane fluorescent staining with DIO.Immunofluorescence double staining was used to labeled spinal PirB in different cell type by cellular markers such as Neu N,GFAP,Iba1 or MBP.Postsynaptic mark synaptophysin and postsynaptic mark PSD95 respectively marked with PirB by double labeling immunofluorescence to determine the position of the spinal PirB at synapses;with the excitatory synaptic markers NR1 and the inhibitory synaptic mark GAD65,PirB was detected in excitatory or inhibitory type of synaptic by double immunofluorescence staining.Real-time PCR was used to detect the m RNA expression of H2-Db and H2-Kb in the spinal cord of SNI mice,and western blot was used to detect the expression of H2-Kb protein.The location of H2-Kb and PirB in the lumbar spinal cord of mice was bipolar labeled by immunofluorescence.The immunoprecipitation protein of spinal PirB in SNI mice were analyzed to explore the downstream mechanism of PirB.Western blot was used to detect the tyrosine phosphorylation of spinal PirB,Trk B and SHP2 after SNI.As signs of excitatory plasticity,the expression of the spinal p-Cam KII,PSD95,NR2 B were detected by Western blot.Mice with spinal PirB overexpression were obtained by intrathecal injection of PirB overexpression plasmid.SNI model was carried on PirB spinal overexpression mice,and the mechanical threshold was measured.The tyrosine phosphorylation levels of spinal PirB,SHP2 and Trk B were detected by Western blot on the 14 th day after SNI,in addition,the expressions of spinal p-Cam KII,PSD95 and NR2 B were detected.The other group of mice with intrathecal injection of PirB overexpression plasmid,were selective ligation of sciatic nerve.Mechanical threshold was observed all the way.At the 7th day after ligation,Trk B receptor agonist 7,8-DHF was intraperitoneal injected once.To explore the downstream targets of phosphorylated PirB,the tyrosine phosphorylation of Trk B and the expression of spinal p-Cam KII,PSD95 and NR2 B were detected by Western blot.Results:PirB expression was significantly upregulated in the corresponding segment of lumbar spinal cord after SNI.PirB was expressed on the neuron surface and axon in spinal cord segment,and was very close to the myelin sheath.PirB was in presynaptic membrane of excitatory synapse.As a natural ligand of PirB,H2-Kb may be involved in the regulation of neuropathic pain.PirB may take a role in regulation of spinal excitatory synaptic plasticity in neuropathic pain by interaction with SHP2 and Trk B.SNI mice with spinal PirB overexpression had an increase tyrosine phosphorylation of spinal PirB and SHP2,but with a decreased tyrosine phosphorylation of spinal Trk B.We found a decrease in expression of spinal p-Cam KII,PSD95 and NR2 B in mice with overexpressed spinal PirB,and the mechanical allodynia induced by SNI was reduced.Intraperitoneal injection of Trk B agonist to the SNI mice with overexpressed spinal PirB,could increase spinal p-Cam KII,PSD95,NR2 B expression,but offset the effect of spinal PirB overexpression on SNI-induced mechanical pain.Conclusions:The expression of PirB in spinal cord was up-regulated in a mouse SNI induced neuropathic pain model.Spinal PirB could play a role in regulation of spinal cord synaptic plasticity by downregulate the expression of p-Cam KII,PSD95,NR2 B in excitatory synapses by interacting with SHP2 and Trk B,and attenuates chronic neuropathic pain in mice induced by SNI.