| Part 1:The role of BDNF/trkB-KCC2 signaling in the spinal dorsal horn in the persistent postoperative pain in ratsObjective:To observe whether the activation of BDNF/trkB-KCC2 signaling in the spinal dorsal horn participates the development of the SMIR(skin/muscle incision and retraction)-induced persistent postoperative pain in rats.Methods:(1)129 Sprague-Dawley rats were equally randomized into 3 groups:normal group,sham group and persistent postoperative pain(SMIR)group.Rats in the normal group underwent no operation;rats in the SMIR group received the skin/muscle incision and retraction(SMIR)surgery to build the persistent postoperative pain model;while rats in the groups that received sham surgery underwent the same procedure without skin/muscle retraction.Animal pain behaviors were assessed at postoperative days(PODs)1,3,7,12,22,and 32 after SMIR(n=8/group).At each time point after the behavioral assessment,rats were randomly sacrificed to isolate the L4-6 spinal cords for western blot(n=5/group/time point),while 5 rats from each group were sacrificed to remove the L4-6 spinal cords for immunofluorescence assay at POD 12.(2)72 rats were equally randomized into 4 groups:sham+saline,sham+TrkB/Fc,SMIR+saline and SMIR+TrkB/Fc.The sham+TrkB/Fc and SMIR+TrkB/Fc groups were intrathecally injected with 5?g TrkB/Fc(Sigma,USA)(dissolved in 10?l saline),while the sham+saline and SMIR+saline groups were intrathecally injected with the same volume of saline.Animal pain behaviors were assessed at PODs 1,3,7,12,22,and 32 after SMIR surgery(n=8/group).At POD 12,rats were sacrificed to remove the L4-6 spinal cord for western blot(n=5/group)and immunofluorescence assay(n=5/group).Results:(1)We found that the MWT in the ipsilateral paw to surgery of SMIR group was significantly and persistent lowered at postoperative days 3-32,which reflected a state of persistent postsurgical pain(P<0.05).Simultaneously,the BDNF and trkB expressions were significantly upregulated in the spinal dorsal horn,while the KCC2 protein were obviously decreased(P<0.05).(2)No significant difference were found between the four groups(P>0.05).Compared with SMIR+saline group,the MWT of SMIR+TrkB/Fc group were significantly increased at postoperative days 7-22,but still lowered than the groups underwent sham surgery(P<0.05).In postoperative day 12,the western blot analysis and immunofluorescence assay both showed evidence of KCC2 upregulation in the spinal dorsal horn of SMIR+TrkB/Fc group compared with SMIR+saline group(P<0.05).However,KCC2 downregulation was only partly reversed and the expression did not return back to the baseline compared with rats underwent sham surgery with saline or TrkB/Fc treatment.Conclusions:The BDNF/trkB-KCC2 signaling in the spinal dorsal horn may participate in the development of SMIR-evoked persistent postoperative pain.Part 2:The effect of dexmedetomidine on the persistent postoperative pain and the BDNF/trkB-KCC2 signaling in the spinal dorsal horn in ratsObjective:To observe the effect of intraperitoneally(IP)injections of dexmedetomidine(DEX)on the SMIR-evoked persistent postoperative pain and the BDNF/trkB-KCC2 signaling in the spinal dorsal horn in rats.Methods:(1)72 rats were randomly and equally divided into 4 groups:sham+saline,sham+DEX,SMIR+saline and SMIR+DEX.The sham+DEX and SMIR+DEX groups were intraperitoneally(IP)injected with 40?g/kg DEX(Jiangsu Hengrui Medicine Co.,Ltd)at 15min pre-surgery and at PODs 1-3,while the sham+saline and SMIR+saline groups were IP injected with the same volume of saline.Pain behaviors were evaluated at pre-surgery day 1 and PODs 1-32(n=8/group).At POD 12,rats were sacrificed to remove the L4-6 spinal cord for western blot(n=5/group)and immunofluorescence assay(n=5/group).(2)16 rats were equally randomized into 2 groups:SMIR+DEX+DMSO group and SMIR+DEX+VU0240551 group.The two groups were both IP injected with 40?g/kg DEX at 15 min pre-surgery and at PODs 1-3;at the same time,rats in the SMIR+DEX+DMSO group were intrathecal ly administered 10?10.01%DMSO,while rats in the SMIR+DEX+VU0240551 group were intrathecally administered 0.27?g VU0240551(Sigma,USA)(dissolved in 10?l 0.01%DMSO).Pain behaviors were evaluated at pre-surgery day 1 and at PODs 1-32(n=8/group).Results:(1)No significant difference were found between the four groups(P>0.05).Compared with SMIR+saline group,the MWT of SMIR+TrkB/Fc group were significantly increased at postoperative days 7-22,but still lowered than the groups underwent sham surgery(P<0.05).In postoperative day 12,the western blot analysis showed that the expressions of BDNF and trkB in the spinal dorsal horn were obviously downregulated,while KCC2 expression was significantly upregulated(P<0.05),and the immunofluorescence assay also exhibited the upregulation of KCC2 in the spinal dorsal horn(P<0.05).(2)Compared with SMIR+DEX+DMSO group,the MWT of SMIR+DEX+VU0240551 group were significantly decreased at postoperative days 1-32(P<0.05).Conclusions:Dexmedetomidine could significantly alleviate the SMIR-evoked persistent postoperative pain,which may work through the modulation on the BDNF/trkB-KCC2 signaling in the spinal dorsal horn. |
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