Stress-Induced Hyperalgesia Of Incision Pain In Female Rat Is Associated With The Descending Facilitation System

Background:Clinical studies find that preoperative stress is a widespread phenomenon.Stress level of women is usually higher,caused the post-operative pain and discomfort feel are also higher.Postoperative hyperalgesia refers to under noxious stimuli the pain sensitivity of body enhanced,which can lead to patient psychological disorder and affect the quality of postoperative recovery.Preoperative stress can increase postoperative pain sensitivity,postoperative analgesic requirements and the suffering of patients.This phenomenon is known as stress-induced hyperalgesia(SIH).Studies found that preoperative psychological distress and postoperative pain sensitivity werea significant positive correlation.The postoperative pain score and the need of analgesic drugs increase for the patients with high stress scores in preoperative stress.Preoperative stress aggravates postoperative hyperalgesia of female patients is a common and easily overlooked phenomenon,which is a major challenge in the field of.postoperative pain research.Since the signaling mechanism of SIH in female patients is not yet completely clear,we study the molecular mechanism have an important significance for clinical treatment programs.Studies of the endogenou pain descending system show that the periaqueductal gray(PAG)and the rostral ventromedial medulla(RVM)play an important role in pain modulation process.Descending inhibitory system and facilitatory system can cause analgesia effect or hyperalgesia by inhibiting or promoting the activity of nociceptive information transfer respectively.Under anxiety state,the expression of G protein-coupled receptor 30(GPR30)in amygdala of female mice increases,which in combination with estrogen can significantly reverse the attenuation trend of GABAA receptor and make it up-regulation.PAG can modulate pain signals through projected regulation of the off-cell,or on-cell neurons within RVM.The PAG and RVM of endogenous pain descending systems can modulate the pain sensitivity information through the spinal dorsolateral fasciculus and ventrolateral fasciculus downward the spinal cord dorsal horn.Recent studies suggest that 5-HT2B receptor,methyl-D-aspartate(NMDA)receptor and PKCy are co-expressed in rat spinal cord neurons.The NR1 phosphorylation in spinal cord dorsal horn neurons may improve the sensitivity to pain stimuli and promote the hyperalgesia.Thus we explore the mechanism between stress-induced hyperalgesia and endogenous pain descending facilitation system in female animals.Purposes:1.Research the influence of preoperative stress to the behavior of postoperative incision pain in female rats;2.Study the role of PAG GPR30 in descending facilitation system for stress-induced hyperalgesia in female rats;3.Reveal the process of stress-induced hyperalgesia in female rats,the role of 5-HT2B in RVM neurons is associated with PAG and spinal dorsal horn neurons in descending facilitation system;4.Study the 5-HT2B/PKCy/p-NR1 signaling pathway in spinal dorsal horn of female rats in descending facilitation system for SIH,providing new ideas for clinical diagnosis and treatment of stress-induced hyperalgesia.Methods:In this study,the ovariectomized female rats were given the same level of estrogen replacement,using forced swim(FS)to establish the preoperative stress model,and the classic incision pain model for the influence of preoperative stress to pain behavior in female rats.After that we implanted cannula into PAG and RVM for microinjection drugs,and intrathecal injection drugs in the animals to observe pain behaviors and signaling pathway of descending facilitation system.Paw Withdrawal Mechanical Threshold(PWMT)and Paw Withdrawal Thermal latency(PWTL)were assessed for hyperalgesia;Western blot and immunofluorescence were analyzed to detect the molecular mechanism.Results:1.Nociceptive thresholds were decreased on PWMT and PWTL in Group S+I compared with Group I at every time point after the incisional surgery.Immunofluorescence analysis showed:GPR30 and GABAA-?4,?1,? were highly expressed in PAG of the rats received FS conditioning and incisional surgery;The increased 5-HT2BR in RVM neurons poses the possibility that this subtype may be important for descending facilitation system compared Group C;We observed that in the spinal dorsal horn the obvious up-regulation expression of 5-HT2BR,PKCy and NMDAR subunit NR1 which were associated with pain behaviors in group S+I under preoperative stress2.Group G1+I contained rats underwent microinjection selective GPR30 agonist G1 and received surgical incision without FS could still increase postoperative hyperalgesia;Group G15+I rats were treated with PAG microinjection GPR30 antagonist G15 after repeated FS conditioning significantly alleviated the hyperalgesia.Western blot analysis showed:GPR30 protein in Group S,Group S+I,Group Gl+I increased,while GABAA-?4,?1,? protein only in Group S+I group and Group G1+I significantly increased;GPR30 and GABAA-?4,?1,?protein expression in Group G15+1 were significantly down-regulated,immunofluorescence results were consistent with Western blot and pain behavior.3.Through RVM microinjection 5-HT2B agonist(BW723C86)and antagonists(SB204741),we found that RVM given BW723C86 induce female rats hyperalgesia,and microinjection of SB204741 could inhibit the hyperalgesia caused by stress.Western blot:5-HT2B protein in Group S+I,Group G1+I and Group BW723C86+I increased;while 5-HT2B protein expression in Group G15+I and Group SB204741+I were significantly down-regulated.5-HT2B receptor expression also could be observed significantly increased in Group BW723C86+I in immunofluorescence,while 5-HT2B receptor expression in Group SB204741+I significantly reduced.4.According to previous research,we gived intrathecal injection 5-HT2B agonists and antagonists,and PKCy inhibitor(C37H65N9013),respectively.The results showed that without preoperative stress,intrathecal injection BW723C86 induced hyperalgesia;intrathecal injection SB204741 could alleviate female rats hyperalgesia after preoperative stress;and intrathecal injection C37H65N9013 suppressed postoperative pain which compared with the control group showed no difference.Western blot analysis showed:5-HT2B expression in Group S+I,Group G1+I,Group BW723C86+I(RVM),Group BW723C86+I(dorsal horn)and Group C37H65N9013+I increased,in Group G15+I,Group SB204741+I(RVM)and Group SB204741+I(dorsal horn)were significantly decreased;PKC?protein expression in Group I,Group S+I,Group Gl+I,Group BW723C86+I(RVM)and Group BW723C86+I(dorsal horn)raised,in Group G15+I,Group SB204741+I(RVM),Group SB204741+I(dorsal horn)and Group C37H65N9013+I decreased;p-NR1 expression in Group S+I,Group G1+I,Group BW723C86+I(RVM)and Group BW723C86+I(dorsal horn)increased,in Group G15+I,Group SB204741+I(RVM),Group SB204741+I(dorsal horn)and Group C37H65N9013+I were significantly reduced.Immunofluorescence showed 5-HT2B receptor expression in Group BW723C86+I was significantly increased and in SB204741+I reduced;PKCy receptor expression in Group C37H65N9013+I also significantly reduced.Conclusion:1.The preoperative stress could induce female rat hyperalgesia significantly in incision pain through endogenous pain descending facilitation system.2.Preoperative stress increased the activation of GPR30 in PAG,which made the GABAA-?4?1? receptor up-regulation.The GABAA-?4?1? subunit changed endogenous descending system function,which suppressed the endogenous descending inhibitory system and promoted the facilitation system function.3.PAG through projection regulated neurons 5-HT2B receptor expression in RVM,further modulation RVM 5-HT projection system to the spinal cord dorsal horn,which induced downstream signaling pathways of 5-HT2B receptor in dorsal horn.4.The increased expression of 5-HT2B/PKC?/p-NR1 signal pathways in spinal cord altered ion channel permeability of NMDA receptor,which increased the sensitivity of neurons to noxious stimuliation in spinal cord and contributed hyperalgesia formation.