Local Structural Changes Around Nodes Of Ranvier In TREZ With Chronic Compression Injury Participate In Orofacial Pain Sensitization In Rats

Background&ObjectivePrimary trigeminal neuralgia（TN）is a paroxysmal and severe chronic neuropathic pain disease.Microvascular compression in the trigeminal root entry zoon（TREZ）,which has a special boundary between the central nervous system（CNS）and the peripheral nervous system（PNS）,is the main cause of TN disease.Whereas the pathogenesis of TN remains unknown.It is found that there is high density of nodes of Ranvier in the CNS-PNS transition band and clearly visible nodes-depleted zones on both sides.And a variety of glial cells participate in the formation of CNS-PNS interface glial barrier.Nodes of Ranvier are important structural bases for the rapid and leaping transmission of electrical signals of myelinated nerve fibers.The neurofascin 155（NF155）at paranodal area can be hydrolyzed by thrombin,causing the dissociation of myelin and the redistribution of ion channels in axons.Paranodal astrocytes can synthesize and secrete endogenous thrombin inhibitors like protease nexin-1（PN-1）participate in the regulating the structure of nodes of Ranvier and neural signaling.Prothrombin can be expressed in small amounts in nervous tissues,but it can also enter the nervous tissue through the blood system from other areas.Immune cells such as macrophages and lymphocytes play an important role in the initiation and maintenance of neuropathic pain sensitization.This study was designed to analyze local structural changes of nodes of Ranvier in TREZ,coagulation-anticoagulant system,blood-nerve barrier permeability and the proportions of immune-related cells in TREZ and TG,to explore the plasticity of nodes of Ranvier,the balance of coagulation-anticoagulation system and the roles of immune-related cells in the pathogenesis of TN.Methods1.Adult male SD rats were randomly divided into chronic compression TN group and sham operation Sham group.The TN rat model was established by retrogradely intubating from the right infraorbital fissure into the skull and compressing the trigeminal nerve root.Von Frey filaments was used to measure the orofacial mechanical stimulation threshold before and after operation.2.To measure the nodal gap length,axon diameter in TREZ and the node-depleted zone length in both sides,immunofluorescence staining was performed with paranodal marker contactin-associated protein（caspr）and myelin basic protein（MBP）while nuclear counterstaining with DAPI.Western Blot methods were used to detect the expression of NF155 and NF125 protein in the TREZ region.3.The transcriptional levels of prothrombin,endogenous thrombin inhibitor PN-1and complement C3 in TREZ region of TN rats were detected by RT-qPCR methods.4.The blood-nerve barrier permeability in TREZ was detected by Evans blue staining methods on post operation 28 days.5.The changes in the numbers and the proportions of lymphocytes,macrophages,microglia,astrocytes in TREZ and TG were detected by flow cytometry methods on post operation 28 days.6.Immunohistochemistry and Western Blot methods were used to detect the expression of thrombin activated receptor（also called protease activated receptor 1,PAR1）in TREZ.Through the exogenous administration of PAR1 receptor inhibitors SCH79797,the effect of PAR1 activation on orofacial hyperalgesia was further studied in TN animal model.Results1.Compared with the Sham group,rats in the TN group developed obvious pain sensitization from POD 14 to 28（p<0.001）.2.During the 7th to 28th day after the operation,the average nodal gap lengths at the CNS-PNS interface in the TREZ in TN group was significantly wider than that in the Sham group（p<0.05）.On the 28th day after operation,the average lengths of the node-depleted zone in CNS and PNS both decreased in TN group comparing with Sham group（p<0.05）.From 7 to 28 days after operation,the expression level of NF155 in TREZ of TN group was higher than that of Sham group（p<0.05）.The expression of NF125 in TREZ in TN group was higher than that of Sham group on the 14th and 21st day after operation（p<0.05）,though there was no significant difference in the expression of NF125between the two groups on the 28th day after operation（p>0.05）.3.The transcription levels of prothrombin,PN-1 and complement C3 in the TREZ were fluctuated at different time points.On the 28th day after surgery,the transcription levels of prothrombin,PN-1 and complement C3 in TREZ up-regulated in TN group（p<0.05）.4.Compared with the Sham group,the exudation of Evans Blue in the TREZ in TN group increased significantly on the 28th day after the operation（p<0.001）.5.Compared with the Sham group,the average volume and granularity of the cells in trigeminal nerve decreased in TN group on the post operation 28 days（p<0.05）.The proportion and the number of CD11b⁺cells in TN group significantly were lower than Sham group（p<0.05）,on the other hand lymphocytes（CD45⁺CD11b^-）and macrophages(CD45^highCD11b⁺)were higher than Sham group（p<0.05）.The proportions and the numbers of microglia(CD45^lowCD11b⁺)in two group had no significant difference（p>0.05）.Compared with the Sham group,the number and proportion of A1 type astrocytes in TN group had a decreasing trend（p=0.159）.And there was no significant difference in astrocytes（CD45^-GLAST⁺）and A2 type astrocytes（CD45^-GLAST⁺S100A10⁺）between two groups（p>0.05）.6.With the Western Blot methods,the expression of PAR1 protein in TREZ in TN group was higher than in the Sham group on the 7th day after the operation（p<0.001）,then it was decreased and recovered to a similar level as the Sham group from the 14th to28th day after the operation（p>0.05）.Immunohistochemical results showed that on the7th day after surgery,PAR1 expression in the TN group was significantly increased,and some of them were co-localized with GFAP immunopositive astrocytes in CNS.There was no significant difference in the distribution of PAR1 expression between the TN group and the Sham group from the 21st to 28th day after the operation（p>0.05）.PAR1receptor inhibitor SCH79797 was injected intraperitoneally one day before the operation and then regularly injected once a week after operation.With the inhibition of PAR1activities,the orofacial mechanical stimulation threshold of rats in TN+SCH79797 group was lower than that of the drug solvent group（TN+DMSO group）on the 3rd and 7th day after surgery（p<0.05）.On the 28th day after the operation,the orofacial mechanical stimulation threshold of rats in TN+SCH79797 group was higher than that of the drug solvent Group（p<0.05）.On the 28th day after surgery,the orofacial mechanical stimulation threshold of rats in TN+SCH79797 group was lower than that in the Sham group（p<0.05）.Conclusions1.After chronic compression injury,the nodal gap in CNS-PNS transitional band was widened,the length of the node-depleted zones in CNS and PNS were both shortened and the glia-axon adhesions also had plastic changes,which indicated that the structure changes of the node of Ranvier had plastic changes.2.After chronic compression injury,the permeability of the blood-nerve barrier changed,the expression of prothrombin,PN-1 and complement C3 increased,and immune-related cells such as glial cells,lymphocytes and macrophages also changed,suggesting that TREZ injurey may induce a local neuroimmune response.3.TREZ chronic compression injury induced plastic changes of the structure of nodes of Ranvier and local neuroimmune microenvironment,which may be involved in the peripheral sensitization process in TN rat animal model.