【作者】 蒋明；

【导师】 顾小萍；

【作者基本信息】 南京大学 ， 麻醉学， 2016， 博士

【摘要】 研究背景:临床研究发现,术前应激状态是一个普遍存在的现象。女性的应激水平通常比较高,手术后疼痛罹患率和不舒适感亦较高。术后痛觉敏化是指术后机体对伤害性刺激产生疼痛敏感性增强的痛觉感受,可导致患者行动或心理障碍,影响患者术后恢复质量。术前应激可以加重患者术后疼痛敏感性,增加术后镇痛药的需要量,这种现象被称为应激导致的痛觉过敏(stress-inducedhyperalgesia,SIH)。研究发现,术前心理困扰与术后疼痛敏感性呈显著正相关,术前应激评分高的患者,术后疼痛评分增高且镇痛药物用量增加。术前应激加重女性患者术后痛觉敏化是一种常见的、易被忽视的现象,也是术后疼痛研究和治疗领域的一大挑战。由于术前应激诱发女性患者术后疼痛敏化的信号机制尚未完全明确,目前没有临床预防和治疗相关的有效方案,因此研究其神经分子机制具有重要意义。对内源性痛觉下行系统的研究表明中央导水管周围灰质(Periaqueductal gray,PAG)和延髓头端腹内侧核(rostral ventromedial medulla,RVM)在疼痛调制过程中作用尤为重要。当下行抑制系统功能增强时表现为镇痛作用,下行易化系统主导时疼痛敏感性增加。研究证实应激状态下小鼠杏仁核中雌激素特异性受体G蛋白偶联受体30(G protein-coupled receptor 30,GPR30)表达增加,与雌激素结合活化的GPR30受体可显著逆转γ-氨基丁酸(γ-Aminobutydc acid,GABA)GABAA受体表达衰减的趋势使其上调。PAG对疼痛的调节可通过对RVM内on-cell、off-cell神经元的投射调节作用调制痛觉信号。RVM经脊髓背外侧束和腹外侧束下行又对脊髓背角神经元的痛觉感受性信息进行调制。近期研究提示在大鼠脊髓背角Ⅱ层神经元内5-HT2B受体和N-甲基-D-天门冬氨酸(N-methyl-D-asparate receptor,NMDAR)受体与核周体包含的 PKCγ 有共表达。NR1亚基磷酸化(p-NR1)增加可提高脊髓背角神经元对疼痛刺激的敏感性,促进痛觉敏化形成。因此内源性痛觉下行易化系统与应激诱导雌性动物术后痛觉敏化的联系机制值得探讨。研究目的:1.研究术前应激状态对雌性切口痛大鼠术后痛行为学的影响;2.明确下行易化系统PAG区GPR30受体对应激引起雌性大鼠术后痛觉敏化的作用;3.揭示应激引起雌性大鼠术后痛觉敏化过程中,下行易化系统RVM区神经元5-HT2B受体与PAG和脊髓背角神经元相关机制作用;4.探讨脊髓背角5-HT2B/PKCγ/p-NR1信号通路在下行易化系统中对雌性大鼠痛觉敏化的作用机制,为临床诊疗应激导致痛觉过敏提供新思路。研究方法:本研究以成年雌性SD大鼠卵巢去势手术后给予同一雌激素水平替代为研究对象,采用强迫游泳(forced swim,FS)建立术前应激模型,并采用切口痛这一经典的术后疼痛模型,研究术前应激状态对雌性大鼠术后痛觉敏化的影响。我们又分别通过PAG区和RVM区置管微量注射药物以及鞘内给药等方法进行干预,观察大鼠疼痛行为和下行易化系统相关信号通路的变化。机械缩足阀值(Paw Withdrawal Mechanical Threshold,PWMT)和热缩足潜伏期(Paw Withdrawal Thermal Latency,PWTL)评定大鼠痛觉敏化行为学;Western blot分析和免疫荧光检测等分子生物学手段探讨相关分子机制。研究结果:1.术前应激后雌性切口痛大鼠术后各时间点PWMT和PWTL较单纯切口痛组大鼠明显降低,其痛行为学可至术后48h。免疫荧光检测显示:PAG内神经元GPR30受体和GABAA-α4、β1、δ受体有所增加;RVM内能神经元5-HT2B亚基较对照组明显增加;术前应激状态可使脊髓内神经元5-HT2B/PKCγ/NR1受体表达增加;2.PAG内置管微量注射GRP30激动剂(G1)且不给予大鼠强迫游泳仍可增加术后痛觉敏化;大鼠完成强迫游泳后微量注射GRP30拮抗剂(G15)则明显缓解术前应激诱发的术后痛敏。Western blot分析显示:S组、S+I组和G1+I组GPR30蛋白表达上调,而GABAA-α4、β1、δ蛋白只在S+I组和G1+I组表达明显增加;在G15+Ⅰ组GPR30和GABAA-α4、β1、δ蛋白表达则明显下调,免疫荧光结果与行为学和Western blot分析结果相一致;3.通过RVM置管和分别微量注射5-HT2B激动剂(BW723C86)及拮抗剂(SB204741)发现,给予RVM区BW723C86可增加雌性大鼠切口痛觉敏化,微量注射SB204741则可抑制应激造成的痛觉过敏。Western blot分析显示:S+I组、G1+I组和BW723C86+Ⅰ组5-HT2B蛋白表达上调;而在G15+Ⅰ组和SB204741+I组5-HT2B蛋白表达则明显下调。免疫荧光同样可以观察到BW723C86+Ⅰ组5-HT2B受体表达上调,SB204741+Ⅰ组5-HT2B受体表达则明显减少;4.根据之前的研究,分别给予大鼠鞘内注射5-HT2B激动剂和拮抗剂以及PKCy抑制剂(C37H65N9013)。结果显示在无术前应激下,鞘内注射BW723C86可诱发切口痛觉过敏;雌性大鼠术前应激造模后鞘内注射SB204741可缓解痛觉过敏,而鞘内注射C37H65N9013则可抑制术后疼痛,与空白对照组相比无差异。Western blot 分析提示:S+I 组、G1+I组、BW723C86+1(RVM)组、BW723C86+I(dorsal horn)组和 C37H65N9013+Ⅰ组脊髓内 5-HT2B 蛋白表达上调,在 G15+1 组、SB204741+Ⅰ(RVM)组和 SB204741+Ⅰ(dorsalhorn)组5-HT2B蛋白表达明显下调:PKCγ蛋白表达在I组、S+I组、G1+I组、BW723C86+I(RVM)组和 BW723C86+I(dorsal horn)组上调,在 G15+1组、SB204741+I(RVM)组、SB204741+Ⅰ(dorsal horn)组和 C37H65N9013+1组表达下调;p-NR1蛋白表达则在S+I组、G1+I组、BW723C86+I(RVM)组和 BW723C86+Ⅰ(dorsal horn)组上调,在 G15+1组、SB204741+I(RVM)组、SB204741+I(dorsal horn)组和C37H65N9013+Ⅰ组表达明显下调。脊髓免疫荧光表达显示BW723C86+Ⅰ组5-HT2B受体表达明显上调,SB204741+Ⅰ组5-HT2B受体表达则明显减少,C37H65N9013+Ⅰ组PKCγ受体表达亦明显减少。研究结论:1.术前应激状态下内源性痛觉下行易化系统可显著诱发雌性切口痛大鼠术后痛觉敏化;2.术前应激使雌性大鼠PAG区GPR30受体活化,后者增加了 GABAA-α4β1δ亚基的表达。GABAA-α4β1δ亚基异常激活后在PAG起始了内源性痛觉下行系统通路的改变,抑制了痛觉下行抑制系统,促进了易化系统功能;3.PAG通过下行易化系统对RVM内神经元的投射调节作用,使RVM神经元5-HT2B受体表达增加,后者与5-HT结合后进一步调制下行至脊髓背角的5-HT投射系统,通过大鼠脊髓背角5-HT2B受体作用促发了下游信号通路;4.脊髓内5-HT2B/PKCγ/p-NR1信号通路表达增加改变了 NMDA受体离子通道的通透性,提高了脊髓神经元对伤害性刺激的敏感性,最终促进了雌性大鼠痛觉敏化的形成。更多还原

【Abstract】 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-α4,β1,δ 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-α4,β1,δ protein only in Group S+I group and Group G1+I significantly increased;GPR30 and GABAA-α4,β1,δprotein 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;PKCγprotein 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-α4β1δ receptor up-regulation.The GABAA-α4β1δ 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/PKCγ/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.更多还原