【作者】 李磊；

【导师】 曹靖；

【作者基本信息】 郑州大学 ， 人体解剖与组织胚胎学， 2024， 博士

【摘要】 背景与目的术后慢性痛是临床常见的疾病且机制不明。据报道大约50%左右的患者遭遇术后切口痛,其中5-10%发展成严重的慢性疼痛,并且发病率逐年升高。围手术期睡眠障碍,是影响术后疼痛慢性化最重要的原因之一。本研究前期发现睡眠剥夺可以增加小鼠丘脑室旁核(Paraventricular thalamic nucleus,PVT)神经元的兴奋性,化学遗传学激活PVT可以减少睡眠时间,并且延长切口痛时程。PVT作为信息传递的中继站主要参与睡眠-觉醒、情绪、恐惧、成瘾和奖赏等行为。前期研究显示PVT接受来自蓝斑核(Locuscoeruleus,LC)酪氨酸羟化酶(Tyrosine hydroxylase,TH)阳性神经元的纤维投射。LC是中枢神经元调控睡眠觉醒的重要脑区之一,然而,LCTH-PVT通路在围手术期睡眠障碍延长术后切口痛时程中的作用目前尚不清楚。丘脑-皮层神经通路参与上行感觉信息的整合和传递。内侧前额叶皮质(Medial prefrontal cortex,mPFC)作为疼痛和情绪控制的中枢,其兴奋性改变在慢性疼痛的发生发展中发挥关键性作用,但其是否参与围手术期睡眠剥夺延长切口痛时程尚不知晓。本研究通过建立睡眠剥夺+切口痛模型,利用痛行为学、EEG-EMG、病毒示踪、免疫荧光染色、化学遗传学、光遗传学和投射性光纤记录等手段,围绕PVT上、下游脑区信息传递和行为学调控,探索围手术期睡眠剥夺延长术后切口痛时程的中枢机制,为术后切口痛的预防和治疗提供新的手段和思路。方法1.建立睡眠剥夺+切口痛模型,通过EEG-EMG监测模型小鼠的睡眠情况,痛行为学检测切口痛时程变化,利用免疫荧光c-Fos染色和光纤记录检测PVT神经元活动信号的变化。2.利用化学遗传学操控PVT神经元兴奋性,通过EEG-EMG监测小鼠睡眠时间的改变,痛行为学检测切口痛时程的变化。3.通过病毒示踪和免疫荧光染色解析LC-PVT通路解剖学联系。利用化学遗传学、光遗传学、投射性光纤记录和痛行为学等方法解析该通路的功能关系以及对围手术期睡眠剥夺+切口痛时程的影响。4.利用病毒示踪和免疫荧光染色解析PVT-mPFC通路解剖学联系。通过化学遗传学、光遗传学、投射性光纤记录和痛行为学等方法解析该通路的功能关系以及对围手术期睡眠剥夺+切口痛时程的影响。5.利用病毒示踪和免疫荧光染色解析LC-PVT-mPFC三级神经通路解剖学联系。通过化学遗传学、光遗传学、投射性光纤记录和痛行为学等手段解析该通路的功能关系以及在围手术期睡眠剥夺+切口痛时程中的作用。结果1.行为学检测发现连续3d睡眠剥夺(6h/d)不改变小鼠基础痛阈值,而连续睡眠剥夺5 d可以降低基础痛阈值,单纯切口组小鼠痛觉阈值于术后第7 d恢复至正常水平。而切口+睡眠剥夺组(Incision+SD)小鼠在术后第7d仍存在痛敏样行为,且PVT中c-Fos表达和谷氨酸浓度均较单纯切口组明显增加。光纤记录发现,外界伤害性刺激可增加PVT谷氨酸能神经元的兴奋性。化学遗传学激活PVT谷氨酸能神经元可增加觉醒时间,并延长切口痛时程。相反,抑制或损毁PVT谷氨酸神经元可缩短Incision+SD组小鼠切口痛时程。2.逆行病毒和顺行突触素病毒示踪均可证实LCTH神经元可以投射到PVT谷氨酸能神经元。免疫荧光染色显示,睡眠剥夺可以增加LC中c-Fos和p-CREB的表达。光遗传激活LCTH-PVTCaMKⅡα通路可以增加PVT谷氨酸能神经元活性,并可导致正常小鼠出现痛觉过敏。相反,光抑制该通路可阻断PVT神经元对外界伤害性刺激的反应,并缓解单纯切口痛模型小鼠的机械痛和冷痛。化学遗传激活LCTH-PVTCaMKⅡα通路可增加小鼠觉醒时间,切口术后连续3 d持续激活该通路可延长切口痛时程。抑制或损毁LCTH-PVTCaMKⅡα通路可显著逆转睡眠剥夺造成术后切口痛的慢性化。3.稀疏标记、顺行跨单突触病毒和逆行跨单突触RV病毒,证实PVT谷氨酸能神经元主要投射到mPFC的GABA能神经元。光遗传学激活PVTCaMKⅡα-mPFC可诱发mPFC中GABA能神经元活性增加。化学遗传学激活mPFCGABA能神经元可诱发痛敏样行为,抑制则可以缓解术后切口痛。化学遗传抑制mPFC谷氨酸能神经元诱发痛敏样行为,而激活可缓解小鼠术后切口痛。化学遗传学激活PVTCaMKⅡα-mPFCGABA通路可以诱发痛觉过敏,并延长切口痛时程;抑制该通路可以缓解切口痛,并缩短Incision+SD组小鼠切口痛时程。4.利用Cre酶依赖的逆行跨单突触RV病毒和Cre酶依赖的突触素病毒证实LCTH-PVTCaMKⅡα-mPFC三级神经通路存在直接突触联系。光遗传激活LCTH-PVTCaMKⅡα通路可以增加mPFC中GABA能神经元的兴奋性,并诱发正常小鼠出现痛觉过敏。利用化学遗传学持续激活LCTH-PVTCaMKⅡα-mPFC通路可延长切口痛时程,相反,持续抑制该通路则可逆转围手术期睡眠剥夺造成的切口痛时程的延长。结论围手术期睡眠剥夺增加PVT和LC神经元的兴奋性。LCTH-PVTCaMKⅡα神经通路通过减少睡眠时间增加切口痛时程;并激活PVTCaMKⅡα-mPFCGABA神经通路进一步增加疼痛程度延长切口痛时程,且存在LCTH-PVTCaMKⅡα-mPFC三级神经通路参与调控围手术期睡眠剥夺延长切口痛时程。本研究为临床围手术期治疗疼痛、调节睡眠尽快恢复正常身心健康提供新的理论依据和干预策略。更多还原

【Abstract】 Background and purposePersistent postsurgical pain is a common clinical disorder with the mechanisms remaining elusive.Approximately 50%of surgical patients suffer from postoperative pain,of whom at least 5～10%have severe chronic pain.Clinical and experimental studies have shown that sleep disturbance-induced stress is a potential catalyst of postsurgical pain.In the perioperative period,sleep disturbance is a critical factor for the development of persistent postsurgical pain.Our preliminary results showed that sleep deprivation(SD)increased the excitability of paraventricular thalamic nucleus(PVT)neurons,and the activation of PVT neurons by chemogenetic method prolonged the duration of postsurgical pain.The PVT,mainly composed of glutamatergic neurons,receives projections from the inhibitory neurons in the brainstem,integrates and transmits information to cortex.The PVT has been identified as a crucial signal integration hub of descending and ascending pathways for a variety of behaviors including circadian rhythm,fear,anxiety,depression,and addiction.The locus coeruleus(LC),the major source of norepinephrine(NE)in central nervous system,is the critical central region for sleep and wakefulness modulation.Previous studies have indicated that PVT receives projections from LCTH neurons.Meanwhile,activation of the LCTH-PVT pathway accelerates awakening from general anesthesia.However,the involvement of the LCTH-PVT pathway in perioperative sleep deprivation-induced prolongation of postsurgical pain remains uncertain.The thalamic-cortical pathway plays a crucial role in integrating and transmitting sensory information.Abundant evidence has shown that PVT projects dense fibers to several cortical regions.The medial prefrontal cortex(mPFC)is the hub for pain and emotion regulation.Previous studies have shown that the PVT-mPFC pathway regulates visceral pain and the depressive-like behaviors induced by chronic pain.However,the relationship between the PVT-mPFC pathway and the perioperative sleep deprivation-induced prolongation of postsurgical pain remains unknown.In this study,a model of postsurgical pain with sleep deprivation(6 h/day for 3 consecutive days)was established,and methods including viral tracing,immunostaining,chemogenetics,optogenetics,fiber photometry,were utilized to investigate the neural circuit mechanism for perioperative sleep deprivation exacerbating postsurgical pain with a specific focus on PVT.These findings offer novel insights and strategies for postsurgical pain management.Methods1.The model of prolonged postsurgical pain was established by plantar incision and sleep deprivation(incision+SD).Electroencephalogram-electromyogram(EEGEMG)recording was used to monitor the sleep patterns.Behavioral tests were used to evaluate the changes in animal behaviors during postsurgical pain.Immunofluorescence staining and fiber photometry were used to detect the activity of the PVT neurons.2.Chemogenetics was utilized to modulate the activity of PVT neurons while behavioral tests were used to evaluate behavioral changes of mice during the postsurgical pain.3.Viral tracing,immunofluorescence staining,chemogenetic,optogenetics,fiber photometry,and behavioral tests were utilized to investigate the anatomical and functional connections of the LC-PVT pathway as well as the role of LC-PVT pathway in the perioperative sleep deprivation-induced prolongation of postsurgical pain.4.To clarify the effect of PVT-mPFC pathway in the perioperative sleep deprivation-induced prolongation of postsurgical pain,viral tracing,immunofluorescence staining,chemogenetics,optogenetics,fiber photometry,and behavioral tests were employed.5.Viral tracing,immunofluorescence staining,chemogenetics,optogenetics,fiber photometry,and behavioral tests were utilized to explore the anatomical connection of the LC-PVT-mPFC tertiary neural pathway and its function in the perioperative sleep deprivation-induced prolongation of postsurgical pain.Results1.Behavioral tests showed that the 3-day sleep deprivation(6 h/d)did not change the physiological pain threshold in na(?)ve mice,while the 5-day sleep deprivation reduced the pain threshold of na(?)ve mice.The pain behaviors of mice in the incision group returned to normal level on day 7,while pain hypersensitivity still presented on day 7 after incision in the mice from incision+SD group.The c-Fos expression and glutamate concentration in PVT were significantly increased in the incision+SD group when compared with those in the incision group.The fiber photometry results showed that the activity of PVTCaMKⅡα+ neurons(PVTCaMKⅡα)increased in response to nociceptive stimuli.Meanwhile,the chemogenetic activation of PVTCaMKⅡα increased the awake time of mice and prolonged the duration of postsurgical pain.In addition,inhibition or ablation of PVTCaMKⅡα eliminated the perioperative sleep deprivationinduced prolongation of postsurgical pain.2.The results of retrograde and anterograde tracing confirmed the projection from LCTH neurons to PVT glutamatergic neurons.The expression of c-Fos and p-CREB in LC was remarkably increased by sleep deprivation.Optogenetic activation of the LCTHPVTCaMKⅡα pathway elevated the activity of PVT glutamatergic neurons and induced pain-like behaviors in na(?)ve mice.On the contrary,optogenetic inhibition of the LCTHPVTCaMKⅡα pathway impeded the response of PVT neurons to nociceptive stimuli and alleviated the incision-induced mechanical hyperalgesia and cold allodynia in mice.In addition,chemogenetic activation of the LCTH-PVTCaMKⅡα pathway increased the awake time of mice.The 3-day consecutive activation of the LCTH-PVTCaMKⅡα pathway by chemogenetics prolonged the duration of postsurgical pain.Inhibition or ablation of the LCTH-PVTCaMKⅡα reversed the prolongation of incision pain induced by sleep deprivation.3.The results of anterograde sparse labeling and anterograde/retrograde transmonosynaptic tracing confirmed the projection from PVT glutaminergic neurons to the GABAergic neurons in mPFC(mPFCGABA).Meanwhile,the mPFCGABA could be activated by the optogenetic activation of the mPFC-projecting PVTCaMKⅡα neurons.Chemogenetic activation of mPFCGABA induced pain-like behaviors in na(?)ve mice,whereas inhibition mPFCGABA alleviated the postsurgical pain of mice in the incision group.On the contrary,Chemogenetic inhibition of the pyramidal neurons in mPFC induced pain-like behaviors in na(?)ve mice,while chemogenetically activating the mPFC pyramidal neurons alleviated the postsurgical pain.Furthermore,activation of the PVTCaMKⅡα-mPFCGABA pathway by chemogenetic methods induced pain-like behaviors in na(?)ve mice and prolonged the postsurgical pain duration of mice in the incision group.Moreover,suppression of this pathway alleviated the postsurgical pain and eliminated the prolongation effect of the perioperative sleep deprivation on the postsurgical pain.4.The application of the anterograde trans-monosynaptic virus and the Credependent synaptophysin virus indicated the exitance of the LCTH-PVTCaMKⅡα-mPFC pathway.Optogenetic activation of the LCTH-PVTCaMKⅡα-mPFC pathway enhanced the activity of mPFCGABA and induced pain-like behaviors in na(?)ve mice.Consecutive activation of the LCTH-PVTCaMKⅡα-mPFC pathway by chemogenetic methods prolonged the duration of postsurgical pain.On the contrary,chemogenetic suppression of this pathway alleviated the postsurgical pain and eliminated the prolongation effect of the perioperative sleep deprivation on the postsurgical pain.ConclusionPerioperative sleep deprivation enhances the neuronal excitability of PVT and LC.The activation of LCTH-PVTCaMKⅡα circuit prolongs the postoperative incision pain by further reducing the sleeping time and activating the PVTCaMKⅡα-mPFCGABA pathway,which increases both of the severity and duration of incision pain.A LCTH-PVTCaMKⅡαmPFC tertiary pathway participates in the perioperative sleep deprivation-induced prolongation of postsurgical incision pain.更多还原