| With the society's development and the speeding up of life rhythm,the important role of psychological factors in the occurrence and development of many diseases has been paid more and more attention.A plenty of researches have proved that psychological stress is related to the occurrence and development of a variety of systemic diseases.At present,the research on the influence of psychological stress on oral and maxillofacial region is clear: the psychological stress can aggravate the occurrence and development of periodontitis,delay the healing of oral mucosal diseases and increase the tension of masticatory muscles.However,as one of the most common oral and maxillofacial diseases,chronic orofacial pain,because of their complex etiology,are affected by many factors.So far,the pathogenesis has not been fully elucidated.In the maxillofacial pain pathway,the primary afferent neurons are located in the trigeminal ganglion,whose peripheral terminals and central terminals are distributed in the face and spinal trigeminal nucleus,respectively.Many studies suggested that the spinal trigeminal nucleus caudal subnuclear(Vc)plays a vital role in the transmission and regulation of orofacial nociceptive information.Based on this,a series of studies have been carried out in our research group.It have been confiremed that astrocytes in the spinal trigeminal nucleus caudal subnuclear as key players in the induction and maintenance of masseter mechanical hyperalgesia induced by psychological stress in rats.Studies have also indicated that spinal trigeminal nucleus caudalis neurons were activated following orofacial nociceptive stimulus,and neuronal glutamate NMDA was involved in the signal transmission between astrocytes and neurons,which played an important role in synaptic transmission.However,it is not clear how NMDA receptors mediate the activation of astrocytes in the process of orofacial pain.In addition,JNK up-regulation in the astrocytes of the spinal cord was found after nerve injury.However,it is not clear how NMDA acts on the activation of JNK under chronic orofacial pain condition.Based on this,we designed the following experiments:Part 1: The establishment and evaluation of animal model of psychological stress and its effects on the masseter mechanical hyperalgesia in rats.First,64 rats were divided into control group,1-day,3-day,5-day,7-day,9-day,11-day and 14-day stress group,each 8 only.Psychological stress was induced by subjecting rats to restraint stress(8h/d),while the rats in the control group were not treated with restraint stress.The results demonstrated that psychological stress could lead to the increased rate of body weight decrease and the negative emotion increase.The results indicated that the chronic restraint stress in this study could result in chronic psychological stress condition of rats.Then,we use Von Frey filaments to testify the mechanical sensitivity of masseter of the rat before and after restraint stress.The results showed that chronic restraint stress could significantly decrease the mechanical pain threshold of rat.The results confirmed the correlation between psychological stress and chronic orofacial pain.Part 2: The role of the NMDA-nNOS-JNK pathway in psychological stress induced rat masseter muscles hyperalgesia.Firstly,effects of psychology factor on the expression of Fos protein and GFAP in the caudal nucleus of the spinal trigeminal nucleus of rats were evaluated by immunofluorescence histochemistry.Results of our study indicated that the expression of Fos protein of rats in the 1-d stress group was significantly increase and then returned gradully to normal level after 1 w,and the vast majority of Fos proteins were expressed in NeuN positive neurons;the expression of GFAP in astrocytes of the Vc was significantly increased in the 7-day,9-day,11-day and 14-day stress groups compared with the control group.These results suggested that restraint stress could effectively activate neurons and astrocytes in the caudal nucleus of the spinal trigeminal nucleus.To further confirm the role of the neurons and astrocytes in Vc in masseter muscles hyperalgesia induced by psychological stress in rats,each stress group was divided into 3 subgroups,c-fos antisense oligodeoxynucleotides(ASO),saline and astrocytic toxin L-?-aminoadipate(L-AA)were intrathecally administrated respectively,one time per day from the first to eleventh day.The rat masseter muscle mechanical allodynia threshold was detected before and after medication in the rats,and then immunohistochemical staining was performed to detect the expression of GFAP and Fos protein in the caudal nucleus of the spinal trigeminal nucleus of rats.The results showed that the two kinds of antagonists can significantly relieve the rat masseter muscle hyperalgesia caused by psychological stress,and c-fos ASO can prevent stress-induced up-regulation of Fos protein and GFAP.Meanwhile,LAA can not only inhibit the expression of GFAP,but also reduce the duration time of the Fos protein expression after stress implement.These results indicated that psychological stress can induce significant activation of Vc neurons and astrocytes,and activation of neurons and astrocytes are closely linked with the development of the mechanical hyperalgesia in master induced by psychological stress in rats through a reciprocal “crosstalk”.Secondly,we investigated the molecular mechanism of the interaction between Vc neurons and astrocytes after psychological stress.In this part,to investigate the activation of the JNK of astrocytes is involved in the process of psychological stress induced masseter muscles hyperalgesia in rats,we used immunofluorescent-histochemistry and Western blotting to detect the expressions of pJNK after the model was successfully established.In addition,to observe the pJNK and GFAP colocalization,double immunofluorescent staining was also applied in the present study.Then,JNK inhibitor was intrathecal injected and change of masseter muscle pain threshold after administration was checked.The results showed that after restraint stress for 14 days,the expression of pJNK was significantly increased,which was completely colocalized with GFAP;Western blotting results also showed that the pJNK-1 expression significantly increased after restraint stress for 14 days,and intrathecally infusing JNK inhibitor can significantly prevent restraint stress-induced masseter mechanical allodynia in rats.These results indicated that the activation of JNK in astrocytes played a key role in the process of masseter muscles hyperalgesia induced by psychological stress in rats.Then,we investigated whether the neuronal NMDA receptor was involved in psychological stress induced masseter muscle hyperalgesia and the activation of astrocytic JNK or not.After the rats were subjected to restraint stress for 14 days,we used double immunofluorescent staining to inspect the colocalization of NeuN and NR2 B,GFAP and NR2 B.Western blotting was used to detect the expression of pNR2 B.Then,the MK-801(namely NMDA receptor receptor blocker),ifenprodil(namely NR2 B selective antagonists)and saline were intrathecally injected to the rats,the change of masseter muscle pain threshold and the expression of pJNK were checked by Western blotting after administration.The results showed marked colocalization of NR2 B and neuronal marker NeuN and the expression level of pNR2 B in the animals of the stressed group obviously exceeded that of the control counterpart.In addition,we also found that the two kinds of inhibitors can not only alleviate the masseter mechanical allodynia,but also significantly inhibit the phosphorylation of JNK after restraint stress.These results suggest that neuronal NMDA receptors are involved in the masseter muscles hyperalgesia induced by psychological stress in rats,and can induce the activation of astrocytic JNK.In order to further study whether NMDA induced JNK activation is dependented on nNOS-GC-cGMP pathway or not,after the rats was constrained for 14 censecutive days,7-NINA,namely selective inhibitor of neuronal nitric oxide synthase(nNOS),and ODQ,namely selective inhibitor of guanylate cyclase(GC)were intrathecally implemented.Then,we checked the change of masseter pain threshold and the expression of pJNK by Western Blotting after administration.The results indicated that intrathecal administration of 7-NINA and ODQ can not only effectively alleviate masseter mechanical allodynia induced by restraint stress,but can also significantly inhibit the expression of pJNK.The results suggested that NMDA receptor activation could increase JNK phosphorylation in astrocytes after restraint stress,which is dependent on the nNOS-GC pathway.Conclusions: 1.Restraint stress can cause a series of stress changes and effectively simulate the state of psychological stress.2.Psychological stress could significantly reduce the masseter mechanical pain threshold of rats,which confirmed the correlation between psychological stress and chronic orofacial pain.3.Psychological stress could lead to the activation of Vc neurons and astrocytes;Inhibition of activation of neurons or astrocytes could reduce mechanical pain in the masseter muscle,while reducing the activaties of the other reciprocally.These results indicate that the activation of neurons and astrocytes are closely related with the rat masseter muscle hyperalgesia induced by psychological stress through a reciprocal “crosstalk”.4.After Psychological stress,the activation of actrocytic JNK of spinal trigeminal nucleus caudalis was significantly enchanced,which was involved in masseter mechanical allodynia induced by psychological stress.Neuronal NMDAR activation was involved in the phosphorylation of astrocytic JNK through a reciprocal “crosstalk”.Phosphorylation of astrocytic JNK induced by psychological stress depends on the nNOS-GC pathway.These results indicated that NMDA-nNOS-JNK pathway play a key role in the process of masseter muscle mechanical hyperalgesia induced by psychological stress in rats. |
PDF Full Text Request