Role Of TLR4 Signal Pathway In Trigeminal Ganglion Involved In The Neural Immune Response Induced By TMJI

Objective: Temporomandibular joint Inflammation(TMJI)induces a degenerative disease characterized by pain,synovitis,progressive degeneration of cartilage and reconstructive of subchondral bone etc.The etiology of TMJI and induced degenerative disease has been considered to be complicated and associated with multiple factors.Studies proved that neurofactors played an important role in the occurrence,development and prognosis of temporomandibular joint(TMJ)infection and followed hyperalgesia.Meanwhile,a variety of cytokines and pain related factors were demonstrated involved in the process of inflammation and pain.Toll-like receptor 4(TLR4)was a pattern recognition receptor involved in many immune and inflammatory diseases.TLR4 signal pathway was gradually become a new research hotspot in neuropathic pain,diabetes,obesity and other diseases.Further studies showed that the expression of TLR4 in primary sensory neurons played an important role in the response to acute injury,maintenance of chronic pain and potential monitoring function.Previous studies in our group showed that macrophages in the trigeminal ganglion(TG)participated in response of immunoreaction and nerve injury.Recent data also showed that TLR4 signal pathway played key roles in the migration and phenotypic alteration of macrophages.Therefore,TLR4 targeting regulation may be effective tools to control macrophages.In this study,we used Freund’s Adjuvant Complete(CFA)to establish a rat model of TMJI and inflammatory pain by intra-articular injection.The dynamic expression of macrophages and TLR4 signal pathway in TG were observed during TMJI,and furthermore to explore the effect and possible mechanisms of TLR4 signal pathway on neural immune response.It was expected to provide a new target for the treatment of TMJI.Materials and Methods: Thirty-three adult male Sprague-Dawley rats were used,provided by Experimental Animal Center of Dalian Medical University.Rats model of TMJI were established by intra-articular injection of CFA as the normal control rats(na?ve group)accepted none treatment.Five Time-Points from 72 hours to 6 weeks post-CFA-treatment were selected for observation and detection,respectively.The rats were perfused by using paraformaldehyde phosphate buffer,and then continuous frozen sections of TG and bilateral TMJ were prepared.While the total RNA in TG were extracted by using the TRIZOL-A+ total RNA extraction procedure for the PCR experimental study.The pathological process in TMJ,protein and gene expression of TLR4,My D88,TRIF,NF-kappa B,TNF-a in TG were detected by HE staining,immunohistochemistry and real time quantitative PCR(RT-q PCR).Results: 1.Histopathological examination showed the progressive pathological changes in the TMJ after CFA injection.At 72 h post-CFA-injection,the synovial hyperplasia and obviously increased synovial folds were detected in TMJ.At 6w post-CFA-injection,chondrocyte disorder,Local fibrocartilage exfoliation and bone defects of condyle were seen.The current results indicated that the TMJI were induced in rats.However,CBCT results showed no obvious bone defect of condyle at 6w post-CFA-injection compared with the na?ve group.2.Immunohistochemical results showed that CD206-labeled M2 macrophages scattered in the TG in both naive and experimental group,and the CD206-positive signals were mainly expressed in the cytoplasm of neurons.The statistical analysis showed that the number of CD206 positive cells per unit area significantly increased from 72 h post-CFA-injection,reached the peak at 2w,continued to 4w post-CFA-injection,and then tended to normal level at 6w post-CFA-injection.3.The results of TLR4 immunohistochemical staining showed that the positive signals of TLR4 were mainly distributed in the cell membrane and cytoplasm of the small and medium-sized sensory neurons in TG.Statistical analysis showed that TLR4-positive neurons increased significantly and reached to the peak at 72 h post-CFA-injection,then tended to normal level at 4w,but increased again at 6w post-CFA-injection.4.The results of My D88 immunohistochemical staining showed that the positive signals of My D88 were mainly distributed in the cell membrane and cytoplasm of the small and medium-sized sensory neurons in TG.The statistical analysis showed that the number of My D88 positive neurons significantly increased from 72h post-CFA-injection,reached the peak at 2w,continued to 4w,and then tended to normal level at 6w post-CFA-injection.5.Correlation analysis for the expression of TLR4 and My D88 in TG showed the low correlation between TLR4 and My D88,r=0.392 which indicated the statistical significance.6.The results of RT-q PCR showed that the expression of TLR4 m RNA in TG significantly increased from 72 h to 6w after CFA treatment(p<0.0001).Meantime,the NF-κB m RNA in TG increased significantly at 72 h and 6w post-CFA-injection,and the TNF-α m RNA increased significantly from 2w to 6w post-CFA-injection.However,TRIF m RNA significantly increased at 6w only(p<0.01).Conclusion: 1.TLR4 signal pathway in TG involved in neural immune response during TMJI.2.The signal transduction of the activated TLR4 in neurons mainly mediated by My D88 dependent pathway in the pathological process of TMJI.3.M2 macrophages in TG involved in the neuroinflammation and repair process during TMJI.