Investigation On The Mechanism Of Morphine-induced Hyperalgesia Through 5-HT_2AR-KCC2 Pathway In Model With Incision Pain

Objective: Postoperative pain is the most common and most urgent pain which needed to deal with immediately.It is the big problem to either doctors or patients.Opioid induced hyperalgesia is one of the side effects of this drug therapy,which is shown to reduce the analgesic effect of the drug,and even to promote pain perception.GABA-A receptor was the main inhibitory receptor of superficial layers of the spinal dorsal horn neurons,whose inhibitory function was mainly affected by the specific expression of the nerve cells-potassium chloride cotransporter（potassium-chloride co-transporter KCC2）.5-HT2 AR can phosphorylate KCC2 to enhance its activity and increase its expression level on the surface of the cell membrane of KCC2 after be activated by PKC.The aim of this study was to reveal the effects of 5-HT_2AR-KCC2 signaling pathway on postoperative pain and morphine induced postoperative pain sensitization.Methods: 1.To prepare postoperative pain model（rat plantar incision pain）,and to observe the role of 5-HT2 AR in the model of pain by intrathecal administration of 5-HT2 AR antagonist and agonist.The MWT and TWL are detected in each group.The role of KCC2 protein in rat spinal dorsal horn neurons was investigated in the animal model.Blot western and RT-PCR technique were used to observe the changes of the transcription and protein levels of the spinal dorsal horn neurons（m RNA and protein）in this model.2.Morphine induced pain sensitization model was made,Compared to DMSO,another two groups are administrated intrathecally by 5-HT2 AR antagonist or agonist to observe the role of 5-HT2 AR in the model of morphine induced pain.The MWT and TWL are detected in each group.The role of KCC2 protein in rat spinal dorsal horn neurons was investigated in the animal model.Blot western technique was used to observe the changes of protein levels of the spinal dorsal horn neurons in the animal model.Results: 1.The role of 5-HT_2AR-KCC2 pathway in postoperative pain 1)Compare to normal rats,the MWT and TWL were significantly decreased of the model of incisional pain.After the intrathecal administration of agonist,MWT were raised obviously while the intrathecal administration of antagonists,MWT were decreased obviously.2)The expression of KCC2 was down regulated.in the incision group continuously.In the day 1 the expression level of KCC2 is also up regulated after agonists were the intrathecal administrated and the expression level of KCC2 is down regulated in the antagonist group.2.The role of 5-HT_2AR-KCC2 signaling pathway in the spinal dorsal horn neurons in pain sensitization induced by morphine 1)Repeated systemic morphine treatment will induce MWT and TWL decreased.The MWT in the rat with intrathecal administration of 5-HT2 AR agonists was higher than those in the control group the day after surgery;Three days after operation,the MWT in the rat with intrathecal administration of 5-HT2 AR antagonist was lower than those in the control group;These results suggest that 5-HT2 AR antagonists may aggravate the pain sensitivity in morphine induced pain in rats,while 5-HT2 AR agonist may be involved in the analgesic effect in the morphine induced pain model.2)The expression of KCC2 in a rat model of incisional pain with repeated systemic morphine treatment was significantly decreased in the third and fifth day after operation compare to the control group.5-HT2 AR receptor agonist TCB-2 significantly increased the expression of KCC2 protein in the spinal cord of rats with morphine induced pain sensitization.The antagonist ketanserin significantly reduced the expression of KCC2 protein in the spinal cord of rats with morphine induced pain sensitization.Conclusion: In the model of incision pain with or without morphine induction,KCC2 is up-regulated by activating 5-HT2 AR receptors.It is suggested that 5-HT_2AR-KCC2 pathway has involved in the mechanism of hyperalgesia.