| Objective Trigeminal neuralgia(TN)is a chronic neuropathic pain with a high incidence in oral and maxillofacial surgery,which seriously affects the quality of life of patients.Although there are many surgical,drug and physical therapy methods for trigeminal neuralgia,they all have a high recurrence rate and are difficult to completely cure the disease.The pathway that pituitary adenylate cyclase activating polypeptide(PACAP)and its specific receptor PAC1 receptor can convert extracellular signal-regulated kinase 1/2(ERK1/2)into its phosphorylation state(p-ERK1/2)and then regulates the sodium channel protein 1.7(Nav1.7)has been verified in guinea pig cardiac neurons,the decreasing of PACAP can reduce the expression level of Nav1.7 through MAPK/ERK pathway in guinea pig cardiac neurons,thereby reducing the excitability of guinea pig cardiac neurons.However,whether this pathway is established in the trigeminal ganglion(TG)of the TN model rat has not been studied.In this study,a TN rat model was established by infraorbital nerve chronic constriction injury(ION-CCI),the difference of mechanical threshold and the expression change of activating transcription factor 3(ATF3)were utilized to verify whether the modeling was successful,and the intrathecal injection of PAC1 receptor antagonists and MAPK/ERK inhibitors were conducted to inhibit the molecular effects of PACAP and its receptor PAC1 receptor and MAPK/ERK pathway.This study utilized above experimental procedures to explore the expression changes and mechanism of PACAP and its receptor PAC1 receptor,MAPK/ERK pathway and Nav1.7 in TG of TN model rats so that can provide new ideas for clinical treatment of TN.Study design 1.Establish a rat model of TN and verify whether the model is successfully produced through behavioral tests and the expression of ATF3Adult male SD rats were randomly divided into ION-CCI group and sham operation group.Chronic constriction of the left infraorbital nerve was performed under aldehyde anesthesia.The operation in the sham operation group was basically the same as that in the ION-CCI group except that the chronic constriction was not performed after the exposure of the left infraorbital nerve.After the surgical modeling,behavioral experiments were used to detect the mechanical thresholds around the vibrissa pads of the ION-CCI group and the sham operation group within 3-42 days after operation,and the differences between the mechanical thresholds of the rats in the two groups were recorded and compared.Rats with positive characteristics in behavioral tests were selected,and q RT-PCR and Western blot experiments were used to detect the expression level of ATF3 in the TG on the operated side to assist in judging whether the rat TN model was successfully established.2.Compare the m RNA and protein expression changes of PACAP,PAC1 receptor,ERK1/2,p-ERK1/2 and Nav1.7 in TG of rats in ION-CCI group and sham operation groupThe expression changes of PACAP,PAC1 receptor,ERK1/2,p-ERK1/2 and Nav1.7 in the operated side TG of rats in ION-CCI group and sham operation group were compared by q RT-PCR and Western blot,and the data were collected and analyzed.3.Explore the molecular mechanism of PACAP,MAPK/ERK pathway and Nav1.7 in TNRats in ION-CCI group were randomly selected and divided into the following three groups according to intrathecal injection of different reagents:(1)PAC1 receptor antagonist PACAP 6-38;(2)MAPK/ERK inhibitor PD098059;(3)normal saline.Rats in each group were administered once a day,and the changes of mechanical threshold around the surgical tentacle pad of rats in each group were detected by behavioral test 1 hour before administration and 1 to 4 hours after administration.The time after administration with the most obvious difference in mechanical threshold was selected,and the PACAP,PAC1 receptor,ERK1/2 p-ERK1/2 and Nav1.7 in the operated side TG of rats in each group were detected by q RT-PCR and Western blot after intrathecal injection of two inhibitors and normal saline.The data was collected and analyzed.Results 1.Compared with the Sham group,the mechanical threshold around the vibrissae pad on the operated side of rats in the ION-CCI group continued to decrease within 3-42 days,and reached the lowest value on the 14 th day.2.The expression of ATF3 in the TG of rats in the ION-CCI group significantly increased.3.After ION-CCI,the expression of PACAP in the TG of rats significantly increased,the expression of ERK1/2 was not significantly different,the expression of p-ERK1/2 significantly increased,and the expression of Nav1.7 and PAC1 receptors decreased.4.Compared with rats in the ION-CCI group injected with normal saline,the mechanical threshold of the rats in the ION-CCI group injected with the PAC1 receptor antagonist PACAP 6-38 increased,the expression of PACAP decreased significantly,the expression of ERK1/2 showed no statistical difference,and the expression of p-ERK1 /2 decreased significantly,the expression of Nav1.7 decreased significantly,while the expression of PAC1 receptor increased.5.Compared with rats in the ION-CCI group injected with normal saline,in the ION-CCI group injected with MAPK/ERK inhibitor PD98059,the mechanical threshold value of rats increased,the expression of PACAP decreased,and the expression of ERK1/2 showed no statistical difference,the expression level of p-ERK1/2 significantly decreased,the expression level of Nav1.7 significantly decreased,and the expression level of PAC1 receptor increased.Conclusion 1.ION-CCI surgery can induce a continuous decrease in the mechanical pain threshold of rats within 3-42 days after operation,and reached the lowest value on the 14 th day.ION-CCI can induce the high expression of ATF3 in the TG of rats;it shows that we produced TN model successfully.2.Intrathecal injection of PAC1 receptor antagonist PACAP6-38 and MAPK/ERK inhibitor PD98059 in rat TG can relieve the hyperalgesia caused by ION-CCI operation in rats.3.Within the TG of rats,antagonism of PAC1 receptor and inhibition of MAPK/ERK pathway can lead to decreased expression of PACAP itself,suggesting that it may be an autocrine regulator.4.In the TG of rats,the expression of PACAP and PAC1 receptor showed an opposite trend.5.The inhibition of PACAP reduces MAPK/ERK pathway activation and Nav1.7 expression,which contributes to the alleviation of ION-CCI-induced hyperalgesia.Conclusion: PACAP/MAPK/ERK/Nav1.7 pathway plays an important role in TN.PACAP is a potential therapeutic target for TN. |
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