Mechanism Of Central Neuroglia Involved In Hyperalgesia And Anxiety-like Behaviors Induced By Tooth Movement

Prevention and treatment of orthodontic patients with pain and stress response such as anxiety and depression is the key to improve patient compliance and obtain fine curative effect.The first is about hyperalgesia caused by orthodontic tooth movement.Studies have shown that spinal astrocytes play a key role in hyperalgesia and pain maintenance.The medullary dorsal horn(MDH)is responsible for the integration of noxious information from the oral and facial region.However,whether astrocytes in MDH participate in orthodontic pain caused by tooth movement and its related mechanism are rarely reported.Therefore,this study aims to investigate the role of MDH astrocytes(AST)in ETMinduced orthodontic hyperalgesia and its detailed mechanism.In this study,we established a model of orthodontic tooth movement,and took MDH as the research target to study the detailed mechanism of hyperalgesia induced by tooth movement by intrathecal administration of FC(AST inhibitor)and IL-1ra(IL-1RI inhibitor).The second is about the anxiety-like behaviors caused by orthodontic tooth movement.Previous studies have shown that minocycline can alleviate anxiety like behavior and cognitive impairment,but the detailed mechanism is still unclear.Interestingly,some studies have shown that cannabinoid receptor II(CB2R)appears to play an immune role similar to minocycline.Therefore,we speculate that CB2 R may be involved in the anti-anxiety and anti-depression effects of minocycline.In order to test this hypothesis,an orthodontic tooth movement model was established,and the hippocampus involved in stress response was taken as the research target.We studied the specific mechanism of anxiety-like behaviors induced by tooth movement through intracerebral cannula administration of Mino and AM630(selective CB2 R antagonist).Part 1.The role of astrocyte activation in orofacial hyperalgesia induced by experimental tooth movement.Objective The study aimed to investigate the involvement of astrocytes in the MDH in the orofacial hyperalgesia induced by ETM and related mechanism.Methods1.The pain threshold(PPT)of the three groups were measured using von Frey filaments from 4 days before ETM operation to 1 day,3 days,5 days,7 days,9 days,11 days and 14 days after ETM operation.2.After behavioral test,frozen sections of MDH area were prepared at each time point,GFAP and c-fos immunofluorescence staining were performed,and GFAP,IL-1β and p-NR1 were analyzed by Western blot.Double immunofluorescence staining of GFAP and IL-1β was performed in ETM group.3.FC was administered intrathecally 3 days after ETM,and then the pain threshold of each group was measured using von Frey filaments.Western-blot and immunofluorescence staining were used to respectively detect GFAP,IL-1β and p-NR1 qualitatively and quantitatively.4.Double fluorescent staining of IL-1RI and p-NR1 was performed in ETM group.Three days after ETM,the rats were intrathecally injected with IL-1R antagonist(IL-1ra),and then the pain threshold was detected.One hour after intrathecal administration of IL-1ra,Western blot analysis of p-NR1 were performed.Results1.The tooth movement distance of ETM group was 166.21 ± 19.22 μm,which was significantly different from that of control group(2.86±0.96 μm)and sham group(3.14±0.23 μm)(P < 0.01).Experimental tooth movement-induced orofacial hyperalgesia from 1to 9 days as the PPT was significantly reduced(p<0.05),with the lowest peak on the third day.2.The results obtained by immunofluorescence analysis was that the expression of c-Fos in MDH was dramatically upregulated at 1 day and 3 days after ETM,while GFAP expression with both immunofluoroscence staining and Western blotting was significantly enhanced at 3 days and 7 days after ETM.Besides,in ETM group,GFAP and IL-1βcoexisted in a large amount.Western blotting analysis indicated that the expression of IL-1β and p-NR1 in MDH was significantly enhanced at 3 days and 7 days after ETM.3.It showed that fluorocitrate administration at 3 days after ETM could markedly suppress the activation of astrocytes(GFAP expression decreased),expression of c-Fos,IL-1β and p-NR1 and attenuate the reduction of PPT induced by ETM.4.Immunofluorescence staining showed that IL-1RI and p-NR1 were co-localized in Neu N positive cells.We found that IL-1ra administration at 3 days after ETM could markedly suppress the expression of p-NR1 and attenuate the reduction of PPT induced by ETM.Conclusion It is the first time that the activation of astrocytes in MDH is associated with hyperalgesia in rats with tooth movement.It may be that astrocyte activation significantly promotes the IL-1β release.IL-1β binds to IL-1RI on neurons,which mediates the phosphorylation of p-NR1 in neurons,improveing the activity of neurons and pain conduction,thus inducing hyperalgesia.Therefore,"astrocyte-IL-1β-(p-NR1)neuron" signaling pathway may be involved in hyperalgesia induced by orthodontic tooth movement.It is suggested that inhibition of astrocyte activation in MDH may be a new way to treat orofacial pain caused by ETM.Part 2.The role of CB2 R in hippocampal microglia in anxiety-like behaviors response induced by ETM.Objective To investigate the role of CB2 R in hippocampal microglia in anxiety-like behaviors induced by ETM.Methods1.In order to observe the weight growth rate of the three groups of animals,the body weights were measured at 1,3,7 and 14 days after operation.ABC-ELISA analysis was used to detect the expression of stress-related hormones in serum of three groups,such as CORT and ACTH.Open field and elevated plus maze behavioral tests were performed.2.Immunofluorescence double labeling technique with Iba-1 and CB2 R antibody were used to determine the cell types involved in the dynamic process in DG of hippocampus 14 days after ETM.The dynamic changes of CB2 R and Iba-1 were observed by quantitative analysis of Western-blot.3.The cannula was placed in bilateral hippocampus by stereotactic operation.After continuous microinjection of minocycline(Mino)and CB2 R antagonist(AM630)into hippocampus for 14 days,the changes of corresponding indexes were detected.They were randomly divided into four groups: sham,14d/ETM,14d/ETM+ Mino and 14d/ETM +AM630 + Mino.Behavioral tests(open field and elevated cross maze)were performed.Immunofluorescence staining and Western blot analysis of Iba-1and CB2 R in DG area of hippocampus were performed.Results1.The weight gain of ETM group was significantly decreased after 7 days and 14 days.The levels of CORT and ACTH of ETM group increased significantly at all time points.Behavior test indicated that ETM group showed obvious anxiety-like behaviors at each time point.2.The immunofluorescence results showed that the microglia in 14d/ETM group were activated like amoeba,and CB2 R and Iba-1 co-located on the surface of microglia 。 However,CB2 R fluorescence labeling and microglia activation were not observed in the control group and sham group.The expression of CB2 R increased significantly in 14d/ETM group by Western-blot analysis.3.Mino prevented the activation of microglia and the up-regulation of CB2 R,and alleviated the anxiety-like behaviors in rats.Compared with 14d/ETM+Mino group,14d/ETM + AM630 + Mino group showed significant anxiety-like behaviors,and the administration of AM630 + Mino failed to inhibit microglia activation and up regulation of CB2 R,indicating that AM630 prevented the effect of minocycline.Conclusion The results showed that the activation of microglia in hippocampus was invoved in the anxiety-like behaviors e induced by tooth movement.And,minocycline could inhibit the activation of microglia and the expression of CB2 R in hippocampus,thus alleviating the a anxiety-like behaviors.However,CB2 R antagonist inhibited the effect of minocycline.Our findings indicate the first time that the activation of CB2 R is required for the anti-anxiety actions of minocycline.This means that CB2 R has become a potential new target for anti-anxiety.