Activation And Polarization Of Macrophages In Trigeminal Ganglia Of Rats

Objective Trigeminal ganglia(TG)play an important role in the process of immune response and regulation of pain induced by maxillofacial injury as it is the location of primary neurons and nociceptors where the oral and maxillofacial sensory afferents and sensory information are integrated preliminarily.Studies showed that macrophages in the ganglia were the first immune cells response to the peripheral inflammation and nerve injury that related to the occurrence and development of hyperalgesia.Macrophages were focused by researchers and considered to be the therapeutic target of many diseases as the play important role in the disease process due to their phenotypic variability and functional diversity.According to the activation state and functions,macrophages were divided into M1 type(classically activated macrophage),which were considered to involve in the inflammatory reaction;M2 type(alternatively activated macrophage),which related to anti-inflammatory reaction,parasite infection,tissue fibrosis and tumor.Therefore,the polarization of macrophages may serve as a regulatory strategy,which provides a new method for the regulation of immune responses in disease.Meanwhile,it should be great significance to study the function of macrophages in different physiological and pathological conditions.In this study,there animal models were established in rats,including pulpitis induced by Complete Freunds Adjuvant(CFA-Pulpitis),inflammation in temporomandibular joint induced by CFA(CFA-TMJ),and chronic constriction injury of infraorbital nerve(CCI-ION).The dynamic expression and distribution of CD68,CD206 positive macrophages in TG were demonstrated.Furthermore,the current study was aimed to investigate the potential mechanism of macrophages that involved in peripheral inflammation and nerve injury,which may provide reference for further clinical therapy.Materials and Methods Adult healthy SPF-Sprague-Dawley rats were used.Three rats were selected randomly as normal control(naive group)and 42 were choose for establishing the animal models,Twelve rats for CFA-Pulpitis group,fifteen for CFA-TMJ group and 15 rats for CCI-ION group.The rats were perfused at different time point from 24 h to 6w post injury(3 rats were used on each time point).Then the bilateral TGs,teeth and TMJ were harvested to make series frozen sections.Dynamic expression and distribution of CD68-,CD206-positive macrophages in TG were demonstrated by immunofluorescence,immunohistochemistry,toluidine blue staining,hematoxylin and eosin(HE)staining.Furthermore,the effects of macrophages involved in the peripheral inflammation,nerve injury and associated hyperalgesia were detected.Results 1.HE staining indicated that the obvious necrotic tissues were being found in the crown of right mandibular molar at 24 hours post CFA-administration,and the necrotic tissues around the exposed point of crown pulp.The typical vacuolar degeneration and angiogenesis were found in dental pulp cavity that indicated the experimental pulpitis was induced.2.HE staining of TMJ revealed pathological changes of TMJ.At 72 h post CFA-inflammation,numbers of synovial folds and angiogenesis were demonstrated and typical fibrinous secretion was observed in the upper cavity of TMJ.After that,chondrocyte clusters were detected in the condyle cartilage.At 6w,the layers of condylar chondrocytes became disordered and subchondral bone were reactive hyperplasia with fibrous layer stripping and condylar defect.The results indicated the inflammation in temporomandibular joint was induced by CFA.3.Neuronal plasticity induced by nerve injury was detected in TG post CCI-ION.Statistical analyses of dynamic expression of calcitonin gene-related peptide(CGRP)indicated that CGRP positive cells were obvious increased at 5 days post CCI-ION.4.Activation of macrophage in TG after CFA induced experimental pulpitis :CD68-positive cells were detected at 24 hours after inflammation.The profiles of the CD68-positive macrophage presented normal rounded morphology with typical extending pseudopodia.At 1 weeks post inflammation,CD68 immunoreactivity macrophages were significantly increased and their pseudopodia were found to extend to form reticular structure among neuronal cells and satellite glial cells in the TG.Till to 6w,a significant reduction of pseudopodia was found.Quantitative analysis of CD68 immunofluorescent density showed that it increased significantly at 72 h and reached the peak at 1w,then reduced and returned to normal level at 6w post inflammation.5.Activation of macrophage in TG after CFA induced inflammation in TMJ :CD68-positive cells were detected at 24 hours after inflammation.At 2 weeks post inflammation,CD68 immunoreactivity macrophages were significantly increased and their pseudopodia were found to extend surrounding neuronal cells in the TG.Till to 6w,a significant reduction of pseudopodia and CD68 immunofluorescent density were found.Quantitative analysis of CD68 immunofluorescent density showed that it increased significantly at 24 h and reached the peak at 2w,then reduced and returned to normal level at 6w post inflammation.6.Activation of macrophage in TG after chronic constriction injury of infraorbital nerve :CD68-positive cells were detected at 24 hours after CCI-ION.The profiles of the CD68-positive macrophage presented a moral rounded morphology with typical extending pseudopodia.At 1 weeks post CCI-ION,CD68 immunoreactivity macrophages were significantly increased and their pseudopodia were found to extend to form reticular structure among neuronal cells and satellite glial cells in the TG.Till to 6w,a significant reduction of pseudopodia was found.Quantitative analysis of CD68 immunofluorescent density showed that it increased significantly at 72 h and reached the peak at 1w,then reduced and returned to normal level at 6w post CCI-ION.7.M2 polarization of macrophage in TG after CFA induced experimental pulpitis: CD206-positive cells were detected at 24 hours after inflammation.The CD206-positive profiles of presented mostly in the cytoplasm.The CD206 positive macrophages appeared normal rounded morphology with rare typical extending pseudopodia.Quantitative analysis of the number of CD206 positive macrophages showed that it increased significantly and reached the peak at 2w,last to 4w and returned to normal level at 6w post inflammation.8.Macrophages in TG participate in the process of pulpitis,peripheral inflammation in TMJ and axonal constriction nerve injury via macrophage activation in TG where the neurons locating and innervating the tooth pulp and TMJ.Conclusion 1.Macrophages in TG participate in the process of pulpitis,inflammation in TMJ and axonal nerve injury via macrophage activation in TG where the neurons locating and innervating the tooth pulp and TMJ.2.Timing of macrophage activation and withdrawal were different in the different course of injury and regeneration.3.M2 polarization of macrophages involve in anti-infection and regeneration process of maxillofacial injury.