The Mechanism Of Epac-1/p120 In The Occurrence And Development Of Persistent Postoperative Pain

Objective To investigate the role and possible mechanism of Epac-1/p120 in persistent postoperative pain.Methods The rat skin/muscle incision and retraction(SMIR)model was established to simulate the clinical operation incision retraction process.The changes of mechanical withdrawal threshold(MWT)were used to evaluate the performance of pain behavior in rats.The leakage of Evans blue solution around the incision was observed;The expression changes of CD34 positive endothelial cells and CD68 positive macrophages in the tissues around the incision after SMIR were observed by immunofluorescence staining and Western blot.The changes of expression of p120 and Epac-1 in tissues around the incision,dorsal root ganglion(DRG)and spinal cord were observed after SMIR;The changes of microglia and astrocytes in spinal cord were also observed.The expressions of inflammatory factors IL-1β and TNF-α in the tissues around the surgical incision,DRG and spinal cord were observed by Western blot.The rats were divided into groups by intraperitoneal injection of 8-pCPT and CE3F4.The changes of pain behavior in each group at different time points were observed.Expression changes of p120 and inflammatory factors IL-1β and TNF-α in the tissues around the surgical incision were detected by Western blot.The expression of CD34 positive endothelial cells and CD68 positive macrophages in the tissues around the incision were detected by immunofluorescence staining.The rats were divided into groups by intrathecal injection of 8-pCPT and CE3F4.The changes of pain behavior at different time points were observed.Expression changes of p120 and inflammatory factors IL-1β and TNF-α in spinal cord were detected by Western blot.The changes of astrocytes in spinal cord were observed by immunofluorescence staining.Results:(1)Compared with the sham group,the rats had progressive pain behavior changes after SMIR,which manifested by the decreased MWT significantly.(2)After operation,the expression of macrophages and endothelial cells in the tissue around the incision increased significantly,and the exudation of Evans blue increased significantly.(3)Compared with the sham group,the expression levels of the inflammatory factors IL-1β and TNF-α in the tissue around the surgical incision,DRG and spinal cord were significantly increased in SMIR group.(4)In the tissues around the incision,Epac-1 was co-labeled with macrophage marker CD68,p120 was co-labeled with both CD68 positive macrophages and CD34 positive endothelial cells;In DRG,Epac-1 was distributed in NF200 positive large neurons and CGRP,IB4 positive small and medium-sized neurons,and p120 was mainly distributed in NF200 positive large neurons;In the spinal cord,Epac-1 was co-labeled with astrocyte marker GFAP,while p120 was co-labeled with astrocyte marker GFAP and a small amount with microglia marker Iba1.(5)After SMIR,microglia in the spinal dorsal horn were activated in the early stage,and then astrocytes were activated,which manifested by the upregulation of the immunoreactivity intensity of Ibal and GFAP in the spinal dorsal horn.(6)Intraperitoneal injection of 8-pCPT can down regulate the level of p120 in the tissues around the incision,increase the expression of inflammatory factors,endothelial cells and macrophages in the tissues around the incision,and significantly promote pain in rats.(7)Intrathecal injection of 8-pCPT could down regulate the level of p120 in the spinal cord and significantly promote pain in rats;It can activate spinal cord astrocytes.(8)After intraperitoneal injection of CE3F4,it can up regulate the level of p120 in the tissue around the incision and reduce the level of inflammatory factors in the tissue around the incision;CE3F4 has obvious analgesic effect on rats after operation.At the same time,it can reduce Evans blue exudation in the tissue around the incision,and significantly reduce the expression of endothelial cells and macrophages.(9)Intrathecal injection of CE3F4 can up regulate the level of p120 in rat spinal cord,reduce the level of spinal inflammatory factors and inhibit the activation of spinal cord astrocytes.Conclusions:Epac-1/p120 is involved in the occurrence and development of persistent postoperative pain.The use of CE3F4 has obvious analgesic effect on rats with persistent postoperative pain,which may be related to the up regulation of p120 level,the inhibition of inflammatory reaction in the tissue around the incision and spinal cord,and the inhibition of the activation of spinal cord astrocytes.