【作者】 李刚；

【导师】 刘斌；

【作者基本信息】 四川大学 ， 麻醉学（专业学位）， 2022， 博士

【摘要】 目的:术后认知功能障碍（Postoperative cognitive dysfunction,POCD）是手术后常见的认知功能损害和中枢神经系统的术后严重并发症,尤其多见于接受手术麻醉的老年病人。POCD的发生率居高不下,可以导致患者术后出现精神、行为和学习能力的下降,造成患者生活质量的明显减低和社会经济医疗负担的加重。因而积极探索POCD的发生机制,寻找行之有效的预防和治疗方法,已成为国际社会普遍关注的热点。虽然目前对POCD发生的病理机制仍然知之甚少,但手术引发的神经炎症在POCD发生机制中的关键作用已得到研究者们的普遍认同。研究发现,在手术引发的神经炎症过程中,中枢炎症因子增加,小胶质细胞活化,凋亡通路的激活都可能参与了炎症损伤导致POCD的中枢机制。研究发现,中枢蓝斑是伤害性神经调节的重要部位,蓝斑神经元及其释放的去甲肾上腺素参与调节神经炎症的发生,蓝斑-去甲肾上腺素系统可能在中枢神经炎性损伤的调控中起着关键作用;在神经退行性疾病中,蓝斑受损变性和去甲肾上腺素水平下降与这些疾病的认知功能障碍发生有关。蓝斑如何调控中枢神经的炎症反应,能否通过调节急性炎症损伤过程中的神经中枢炎症从而达到减轻术后认知功能障碍的目的?蓝斑上存在着丰富的α₂-肾上腺素能受体。已知,一种临床常用的麻醉辅助药--选择性的α₂-肾上腺素受体激动剂右美托咪定能够作用于蓝斑上的α₂-肾上腺素能受体。有研究发现右美托咪定能减少POCD的发生。右美托咪定是否通过激动蓝斑上的α₂-肾上腺素受体,减轻中枢炎症反应,从而发挥抗POCD的作用?目前尚不清楚。为了进一步探索POCD的发生机制,寻找行之有效的预防和治疗POCD方法,本课题以小鼠肠缺血/再灌注模型为研究工具,运用实验药理学的方法,观察α₂-肾上腺素能受体激动剂右美托咪定对肠缺血/再灌注损伤引起的小鼠POCD的防治作用,以及选择性蓝斑去甲肾上腺素能神经毒素DSP-4[N-（2-氯乙基）-N-乙基-2-溴苯胺盐酸盐]或α₂-肾上腺素受体阻断剂阿替美唑与育亨宾对右美托咪定抗小鼠POCD作用的影响;采用免疫组织化学和生物化学方法,探索海马区域炎症因子、小胶质细胞密度、凋亡蛋白表达与其POCD改变的相关性,分析右美托咪定对肠缺血/再灌注损伤引起的POCD小鼠蓝斑核中酪氨酸羟化酶阳性神经元和海马区去甲肾上腺素水平的变化。旨在揭示中枢α₂-肾上腺素能受体激动剂的抗实验性POCD作用,探讨蓝斑去甲肾上腺素系统在抑制中枢炎症反应,发挥神经保护作用,调控POCD中的作用及其机制。希望通过本研究为术后急性神经炎症和认知功能障碍的防治提供新的线索与方法。材料和方法:第一部分:实验将雄性C57BL/6J小鼠72只,随机分为6组,包括对照组（CON）,假手术组（SHAM）,肠缺血/再灌注组（I/R）,肠缺血/再灌注前右美托咪定预处理组（DEX+I/R）,肠缺血/再灌注前DSP-4预处理组（DSP-4+I/R）,肠缺血/再灌注前先后使用DSP-4和右美托咪定预处理组（DSP-4+DEX+I/R）。采用戊巴比妥钠麻醉小鼠,开腹夹闭肠系膜上动脉45min,复制肠缺血/再灌注损伤模型。建模前30min单独使用右美托咪定50μg/kg腹腔注射预处理,或者神经毒素DSP-4 50mg/kg分别于术前14天和7天两次腹腔注射进行预处理,或者先使用DSP-4 50mg/kg分别于术前14天和7天两次腹腔注射后,于建模前30min再给予右美托咪定50μg/kg腹腔注射给药治疗。肠缺血/再灌注24h后,使用Morris水迷宫评估小鼠的记忆功能;用酶联免疫吸附法（ELISA）检测血清和海马区白介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、去甲肾上腺素（NE）和丙二醛（MDA）水平;分别采用免疫荧光测定小鼠蓝斑酪氨酸羟化酶（TH）阳性神经元的表达;免疫组织化学方法评估海马小胶质细胞密度改变;HE染色方法观察缺血引起的肠粘膜损伤,Chiu’s评分评估其损伤程度;用western blot法测定海马区凋亡相关蛋白Bcl-2,Caspase 3,Bax的变化。第二部分:雄性C57BL/6J小鼠60只,随机分为5组,包括SHAM组,I/R组,DEX+I/R组,肠缺血/再灌注之前先后使用阿替美唑和右美托咪定预处理组（ATI+DEX+I/R）,肠缺血/再灌注之前先后使用育亨宾和右美托咪定预处理组（YOH+DEX+I/R）。两种α₂-肾上腺素能受体阻断剂阿替美唑500μg/kg或育亨宾2mg/kg分别在右美托咪定给药之前30min腹腔注射给药;通过ELISA法观察右美托咪定对肠缺血/再灌注损伤后IL-1β、TNF-α、MDA以及NE水平的影响,采用western blot法测定海马区凋亡相关蛋白Bcl-2,Caspase 3,Bax的变化,并通过水迷宫实验观察右美托咪定对肠缺血/再灌注损伤术后早期认知功能的影响。结果:肠缺血/再灌注24h后,小鼠肠粘膜损伤Chiu’s评分明显升高。与SHAM组比较,I/R组小鼠肠粘膜损伤明显（P<0.01）;与I/R组相比,DSP-4+I/R组并没有减轻肠缺血/再灌注引起的肠粘膜损伤,Chiu’s评分无显著性差异（P=0.9992）;反之,肠缺血/再灌注前用右美托咪定预处理却可以明显改善肠粘膜损伤,降低Chiu’s评分;与I/R组相比,使用右美托咪定预处理的DEX+I/R组和DSP-4+DEX+I/R组Chiu’s评分均明显降低（P<0.01）;与DSP-4+I/R组相比,使用右美托咪定预处理的DSP-4+DEX+I/R组Chiu’s评分也明显下降（P<0.01）。表明选择性α₂-肾上腺素受体激动剂右美托咪定能降低缺血/再灌注引起的肠粘膜损伤。水迷宫实验结果发现,肠缺血/再灌注24h后,6个组之间目标平台跨越次数和在目标象限停留的时间均存在明显的统计学差异（P<0.01）。与CON组或SHAM组相比,I/R组小鼠跨越目标平台次数和靶象限停留时间明显减少（P<0.01）;与I/R组相比,右美托咪定预处理DEX+I/R组可以增加小鼠跨越目标平台次数和靶象限停留时间,减轻肠缺血/再灌注损伤所导致的记忆损害（p<0.01）;与I/R组相比,DSP-4+I/R和DSP-4+DEX+I/R组小鼠跨越目标平台次数明显增加,在靶象限内停留时间也明显延长（P<0.01）。证明肠缺血/再灌注损伤可引起小鼠明显的记忆损害;采用蓝斑α₂-肾上腺素受体激动剂右美托咪定预处理或DSP-4预处理毁损蓝斑去甲肾上腺素神经元均能明显减轻肠缺血小鼠术后的记忆功能损害。使用α₂-肾上腺素能受体阻断剂阿替美唑或育亨宾后,与DEX+I/R组相比,ATI+DEX+I/R和YOH+DEX+I/R组小鼠跨越目标平台次数都明显减少（P<0.01）,靶象限停留时间也明显缩短（P<0.01）。提示拮抗α₂-肾上腺素能受体削弱了右美托咪定的认知功能保护作用。小鼠脑切片免疫荧光染色结果显示,肠缺血/再灌注损伤24 h后,6个组之间蓝斑核中TH阳性细胞密度差异具有统计学意义（P<0.01）。与CON组或SHAM组相比,I/R组蓝斑中TH阳性细胞密度显著升高（P<0.01）,但与I/R组相比,使用右美托咪定或DSP-4预处理的DEX+I/R组、DSP-4+I/R组和DSP-4+DEX+I/R组蓝斑中TH阳性细胞密度却明显降低（P<0.01）。6个组之间的NE水平在海马或血清中均存在显著的统计学差异（P<0.01）。与SHAM组比较,I/R组小鼠海马和血清中NE水平均显著升高（P<0.01）;与I/R组比较,血清NE水平在DEX+I/R组明显下降（P<0.05）,而与I/R组比较,血清NE水平在DSP-4+I/R组和DSP-4+DEX+I/R组也显著下降（P<0.01）。证明肠缺血/再灌注损伤能兴奋蓝斑肾上腺素能神经元,从而增加中枢和外周NE的释放;右美托咪定激动蓝斑α₂-肾上腺素受体或DSP-4预处理毁损蓝斑肾上腺素能神经元均能够降低海马和血清中的NE水平。海马和血清样本ELISA分析发现,肠缺血/再灌注损伤24h后,各个组之间海马和血清中IL-1β、TNF-α和MDA均存在明显的统计学差异（P<0.01）。与SHAM组相比,I/R组小鼠海马和血清中IL-1β、TNF-α和MDA均显著升高（P<0.01）,证实肠缺血/再灌注损伤能升高外周血中炎症因子和增加中枢神经系统的炎症反应。在海马组织中,与I/R组比较,IL-1β在DEX+I/R组明显降低（P<0.05）,在DSP-4+I/R组和DSP-4+DEX+I/R组也显著降低（P<0.01）。与I/R组比较,TNF-α在DEX+I/R组,DSP-4+I/R组和DSP-4+DEX+I/R组均显著下降（P<0.01）。与I/R组比较,MDA在DEX+I/R组水平明显减低（P<0.05）,在DSP-4+I/R组和DSP-4+DEX+I/R组也显著降低（P<0.01）。证明右美托咪定或DSP-4预处理均能减轻肠缺血/再灌注引起的中枢神经系统炎症反应。在血清中,与I/R组比较,IL-1β在DSP-4+I/R组显著降低（P<0.01）。与I/R组比较,TNF-α在DEX+I/R组和DSP-4+DEX+I/R组明显减低（P<0.05）,并且在DSP-4+I/R组也显著下降（P<0.01）。与I/R组比较,MDA在DEX+I/R组,DSP-4+I/R组和DSP-4+DEX+I/R组均显著下降（P<0.01）。表明用右美托咪定激动蓝斑α₂-肾上腺素受体或DSP-4预处理毁损蓝斑去甲肾上腺素能神经元,均能减轻外周的炎症损伤。采用对α₂-肾上腺素能受体阻断剂阿替美唑或育亨宾预处理后各组海马样本的ELISA分析发现,肠缺血/再灌注损伤24h后各组之间海马组织中IL-1β、TNF-α、MDA和NE水平均存在显著的统计学差异（P<0.01）。与DEX+I/R组相比,IL-1β在ATI+DEX+I/R组显著升高（P<0.01）,在YOH+DEX+I/R组也显著升高（P<0.05）;与DEX+I/R组相比,TNF-α在ATI+DEX+I/R组和YOH+DEX+I/R组均显著升高（P<0.05）;与DEX+I/R组相比,NE水平在YOH+DEX+I/R组显著升高（P<0.05）;与DEX+I/R组相比,MDA在ATI+DEX+I/R组显著升高（P<0.05）,在YOH+DEX+I/R组也显著升高（P<0.01）。提示选择性α₂-肾上腺素能受体阻断剂阿替美唑或育亨宾阻滞蓝斑去甲肾上腺素系统,拮抗了右美托咪定的中枢抗炎效应。免疫组织化学研究结果显示,各组之间海马CA1区Iba-1阳性细胞密度差异有统计学意义（P<0.01）。与CON组和SHAM组比较,I/R组Iba-1阳性细胞密度明显增加（P<0.01）;与I/R组相比,DEX+I/R组、DSP-4+I/R组和DSP-4+DEX+I/R组海马Iba-1阳性细胞密度显著降低（P<0.01）。表明右美托咪定或DSP-4预处理均能减少肠缺血/再灌注损伤后中枢小胶质细胞的集聚。结论:1、肠缺血/再灌注损伤能增加蓝斑核酪氨酸羟化酶阳性神经元的表达,促进去甲肾上腺素向海马区的释放,加重术后记忆功能的损害。2、选择性α₂-肾上腺素受体激动剂右美托咪定不仅能减轻缺血/再灌注引起的肠粘膜损伤,降低chiu’s评分,而且可减轻术后记忆功能的损害。3、α₂-肾上腺素受体阻断剂阿替美唑和育亨宾则能拮抗右美托咪定的认知功能保护效应,证明α₂-肾上腺素受体参与了认知功能的保护机制。4、采用DSP-4毁损蓝斑肾上腺素能神经元,能抑制去甲肾上腺素的释放,降低脑内IL-1β、TNF-α、MDA含量,减少术后记忆功能损害的发生。提示蓝斑-去甲肾上腺素系统参与中枢炎症反应,调节术后认知功能。5、肠缺血/再灌注损伤能增加脑内的IL-1β、TNF-α和MDA含量,提高海马Iba-1阳性小胶质细胞的密度,表明中枢神经炎症与术后认知功能障碍的发生有关。6、右美托咪定预处理能降低脑内IL-1β、TNF-α和MDA的含量,降低海马Iba-1阳性小胶质细胞密度,减轻术后记忆功能损害,说明中枢α₂-肾上腺素受体激动剂具有抗炎和减轻神经功能损害的作用。综上所述,肠缺血/再灌注损伤不仅能引起全身炎症反应,而且能引发中枢神经系统炎症,能明显增加蓝斑肾上腺素能神经元中酪氨酸羟化酶的表达,促进中枢去甲肾上腺素的释放,引起术后认知功能障碍的发生;毁损蓝斑肾上腺素能神经元能抑制脑内炎症反应,减少术后记忆功能损害;中枢α₂-肾上腺素受体激动剂可通过兴奋蓝斑α₂受体,抑制肾上腺素能神经元的激活,明显改善小鼠肠缺血/再灌注损伤引起的术后早期认知功能障碍;相反,α₂-肾上腺素受体阻断剂阿替美唑和育亨宾则能拮抗其认知功能保护作用。表明蓝斑α₂-肾上腺素受体的兴奋,能反馈性地减少中枢去甲肾上腺素的释放,抑制炎症反应,防止神经功能损伤,减少术后认知功能障碍的发生。更多还原

【Abstract】 Objective:Postoperative cognitive dysfunction（POCD）is a common cognitive impairment after surgery and one of the postoperative complications of the central nervous system,particularly in elderly patients undergoing anesthesia.The high incidence of POCD can lead to the decline of patients’mental,behavioral and learning ability after surgery,resulting in significantly declined quality of life of patients and increased social,economic and medical burden.Therefore,actively exploring the mechanism of POCD and seeking effective prevention and treatment methods have become a hot spot of universal concern in the international community.Although the pathogenesis of POCD is still poorly understood,the key role of neuroinflammation induced by surgery in the pathogenesis of POCD has been widely recognized by researchers.It has been found that during neuroinflammation induced by surgery,the increase of inflammatory factors in central nervous system,activation of microglia and activation of apoptotic pathways may be involved in the mechanism of inflammatory damage leading to POCD.It has been found that central locus coeruleus is an important nuclei of nociceptive neuroregulation in the central nervous system,and locus ceruleus neurons and norepinephrine are involved in regulating the occurrence of neuroinflammation.Locus ceruleus-norepinephrine system may play a key role in the regulation of central nervous inflammatory injury;in neurodegenerative diseases,locus ceruleus damage and decrease of norepinephrine levels are related to the occurrence of cognitive dysfunction in these diseases.How does locus coeruleus regulate the inflammatory response of central nervous system?Can locus coeruleus regulate the inflammation of central nervous system in the process of acute inflammatory injury so as to reduce postoperative cognitive dysfunction?There are abundantα₂-adrenergic receptors in locus ceruleus.It is known that dexmetomidine,a selectiveα₂-adrenoceptor agonist,can activeα₂-adrenergic receptors in locus ceruleus.Some studies have found that dexmetomidine can reduce the incidence of POCD.Does dexmedetomidine play an anti-POCD effect by activatingα₂-adrenoceptor in locus ceruleus to reduce central inflammation?It’s not clear yet.In order to further explore the mechanism of POCD and find effective ways to prevent and treat POCD,in this study we took the mouse intestinal ischemia/reperfusion model as the research tool,and observed the prevention and treatment effect of dexmedetomidine on POCD induced by intestinal ischemia/reperfusion injury in mice by the method of experimental pharmacology.We also observed the effect of selective norepinephrine neurotoxin DSP-4[N-（2-chloroethyl）-N-ethyl-2-bromobenzylamine hydrochloride]orα₂-adrenergic receptor blockers atipamezole and Yohimbine on the prevention and treatment of dexmedetomidine against POCD in mice;Immunohistochemical and biochemical methods were used to explore the correlation between hippocampal inflammatory factors,microglial cell density and apoptotic protein expression and their changes in POCD,and to analyze the changes of tyrosine hydroxylase positive neurons in locus coeruleus and norepinephrine levels in hippocampus of POCD mice induced by intestinal ischemia/reperfusion injury.Findings in this study revealed the anti-experimental POCD effect of dexmedetomidine,and explored the correlation between locus coeruleus norepinephrine system and central anti-inflammatory and neuroprotective effects,which is expected to provide new clues and methods for the prevention and treatment of postoperative acute neuroinflammation and cognitive dysfunction.Materials and Methods:Part Ⅰ:Seventy-two male C57BL/6J mice were randomly divided into 6 groups,including control group（CON）,sham operation group（SHAM）,intestinal ischemia/reperfusion group（I/R）,dexmedetomidine preconditioning group before intestinal ischemia/reperfusion（DEX+I/R）,DSP-4 preconditioning group before intestinal ischemia/reperfusion（DSP-4+I/R）,the group of DSP-4 and dexmedetomidine pretreated before intestinal ischemia/reperfusion（DSP-4+DEX+I/R）.The mice were anesthetized with pentobarbital sodium;the superior mesenteric artery was clamped for 45 min to induce intestinal ischemia reperfusion injury.Dexmedetomidine alone or combined with DSP-4,a selective locus coeruleus noradrenergic neurotoxin,was used for pretreatment.Dexmedetomidine 50μg/kg was administed intraperitoneally 30min before the operation,or DSP-4 50mg/kg was given intraperitoneally twice 14 days and 7 days before the operation.Or DSP-4 50mg/kg was first injected intraperitoneally 14 days and 7 days before the operation,then dexmedetomidine 50μg/kg was injected intraperitoneally 30 minutes before the operation.Morris water maze was used to evaluate the memory function of mice after intestinal ischemia/reperfusion for 24h.The levels of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,norepinephrine（NE）and Malondialdehyde（MDA）in serum and hippocampal were determined by enzyme-linked immunosorbent assay（ELISA）.Immunofluorescence was used to detect the expression of TH positive neurons in mice.The change of microglial density in hippocampus was evaluated by immunohistochemistry.HE staining was used to observe the intestinal mucosal injury caused by ischemia,and Chiu’s score was used to evaluate the degree of intestinal mucosal injury.The changes of apoptosis-related proteins Bcl-2,Caspase3 and Bax in hippocampus were determined by western blot analysis.Part Two:Sixty male C57BL/6J mice were randomly divided into 5 groups,including SHAM group,I/R group,DEX+I/R group,and the group pretreated with atipamezole and dexmedetomidine before intestinal ischemia/reperfusion（ATI+DEX+I/R）.The group pretreated with yohimbine and dexmedetomidine before intestinal ischemia/reperfusion（YOH+DEX+I/R）.Twoα₂-adrenergic receptor antagonists,atipamezole at 500μg/kg or yohimbine at 2mg/kg,were intraperitoneally injected 30min before dexmedetomidine administration,respectively.The effects of dexmedetomidine on the levels of IL-1β,TNF-α,MDA and NE after intestinal ischemia/reperfusion injury were observed by ELISA.The changes of apoptosis-related proteins Bcl-2,Caspase3 and Bax in hippocampus were measured by western blot.The effects of dexmedetomidine on early cognitive function after intestinal ischemia/reperfusion injury were observed by Morris water maze.Results:After intestinal ischemia/reperfusion for 24h,Chiu’s score of intestinal mucosal injury in mice was significantly increased.Compared with SHAM group,intestinal mucosal injury was significant in I/R group（P<0.01）.Compared with I/R group,DSP-4+I/R group did not reduce intestinal mucosal injury caused by intestinal ischemia/reperfusion,and Chiu’s score had no significant difference（P=0.9992）.On the contrary,dexmedetomidine pretreatment before intestinal ischemia/reperfusion significantly improved intestinal mucosal injury and decreased Chiu’s score.Compared with I/R group,the Chiu’s score in DEX+I/R group and DSP-4+DEX+I/R group pretreated with dexmedetomidine was significantly decreased（P<0.01）.Compared with DSP-4+I/R group,the Chiu’s score in DSP-4+DEX+I/R group pretreated with dexmedetomidine was also significantly decreased（P<0.01）.The results indicated that the selectiveα₂-adrenergic receptor agonist dexmedetomidine can reduce intestinal mucosal injury induced by ischemia/reperfusion.The results of the Morris water maze showed that there was a significant difference in the number of target platform crossing and in the time of staying in the target quadrant between the 6 groups after intestinal ischemia/reperfusion for 24h（P<0.01）.Compared with CON group or SHAM group,the number of crossing target platform and the time of staying in the target quadrant of mice in I/R group were significantly reduced（P<0.01）.Compared with I/R group,DEX+I/R group could increase the number of mice crossing the target platform and the time of staying in the target quadrant,and reduce the memory impairment caused by intestinal ischemia/reperfusion injury（P<0.01）.Compared with the I/R group,in the DSP-4+I/R and DSP-4+DEX+I/R groups,the number of mice crossing the target platform was significantly increased,and the time of staying in the target quadrant was also significantly extended（P<0.01）.It was proved that intestinal ischemia/reperfusion injury can cause significant memory impairment in mice.Pretreatment with dexmedetomidine,an agonist ofα₂-adrenergic receptor in locus coeruleus or pretreatment with DSP-4,an agent of destruction the adrenergic neurons in locus coeruleus can significantly reduce postoperative memory impairment in mice with intestinal ischemia.Compared with the DEX+I/R group,the frequency of crossing the target platform in ATI+DEX+I/R and YOH+DEX+I/R groups was significantly reduced（P<0.01）,and the time of staying in the target quadrant was also significantly shortened（P<0.01）after the administration ofα₂-adrenergic receptor blockers atipamezole or yohimbine.It was suggested thatα₂-adrenergic antagonist could weaken the protective effect of dexmedetomidine on cognitive function.The results of immunofluorescence staining of mouse brain sections showed that the density of TH-positive cells in the locus coeruleus of the 6 groups was significantly different 24h after intestinal ischemia/reperfusion injury（P<0.01）.Compared with CON group or SHAM group,the density of TH-positive cells in the I/R group was significantly increased（P<0.01）,but compared with the I/R group,the density of TH positive cells in the locus coeruleus of DEX+I/R group,DSP-4+I/R group and DSP-4+DEX+I/R group was significantly decreased（P<0.01）after dexmedetomidine or DSP-4 pretreatment.There were statistically significant differences in NE levels in hippocampus or serum among the 6 groups（P<0.01）.Compared with SHAM group,the levels of NE in the hippocampus and serum of mice in I/R group were significantly increased（P<0.01）.Compared with the I/R group,the serum NE level in DEX+I/R group was significantly decreased（P<0.05）,and the serum NE level in DSP-4+I/R group and DSP-4+DEX+I/R group was also significantly decreased（P<0.01）.It was demonstrated that intestinal ischemia/reperfusion injury can excite the adrenergic neurons of the locus coeruleus,thereby increasing the release of central and peripheral NE.Dexmedetomidine stimulated theα₂-adrenergic receptor of the locus coeruleus or DSP-4 destroyed the adrenergic neurons of the locus coeruleus,which decreased the level of NE in the hippocampus and serum.ELISA analysis of hippocampal and serum samples showed that there were significant differences in IL-1β,TNF-αand MDA in hippocampal and serum 24h after intestinal ischemia/reperfusion injury among all groups（P<0.01）.Compared with SHAM group,the levels of IL-1β,TNF-αand MDA in hippocampus and serum of mice in I/R group were significantly increased（P<0.01）,indicating that intestinal ischemia/reperfusion injury can increase the inflammatory factors in peripheral blood and increase the inflammatory response of central nervous system.In the hippocampus,IL-1βwas significantly decreased in DEX+I/R group compared with I/R group（P<0.05）,and also significantly decreased in DSP-4+I/R group and DSP-4+DEX+I/R group（P<0.01）.Compared with I/R group,TNF-αwas significantly decreased in DEX+I/R group,DSP-4+I/R group and DSP-4+DEX+I/R group（P<0.01）.Compared with I/R group,MDA level in DEX+I/R group was significantly decreased（P<0.05）,and also significantly decreased in DSP-4+I/R group and DSP-4+DEX+I/R group（P<0.01）.Both dexmedetomidine and DSP-4 preconditioning were shown to reduce neuroinflammation in central nervous system induced by intestinal ischemia/reperfusion.In serum,IL-1βwas significantly decreased in DSP-4+I/R group compared with I/R group（P<0.01）.Compared with I/R group,TNF-αwas significantly decreased in DEX+I/R group and DSP-4+DEX+I/R group（P<0.05）,and also significantly decreased in DSP-4+I/R group（P<0.01）.Compared with I/R group,MDA was significantly decreased in DEX+I/R group,DSP-4+I/R group and DSP-4+DEX+I/R group（P<0.01）.The results showed that dexmedetomidine activatedα₂-adrenergic receptor in locus coeruleus or DSP-4 pretreatment damaged adrenergic neurons in locus coeruleus,which could reduce the peripheral inflammatory injury.ELISA analysis of the hippocampal samples of each group pretreated withα₂-adrenergic receptor blocker atipamezole or yohimbine showed that there were significant differences in the levels of IL-1β,TNF-α,MDA and NE in the hippocampal tissues of all groups 24h after intestinal ischemia/reperfusion injury（P<0.01）.Compared with DEX+I/R group,IL-1βwas significantly increased in ATI+DEX+I/R group（P<0.01）and YOH+DEX+I/R group（P<0.05）.Compared with DEX+I/R group,TNF-αwas significantly increased in ATI+DEX+I/R group and YOH+DEX+I/R group（P<0.05）.Compared with DEX+I/R group,NE level in YOH+DEX+I/R group was significantly increased（P<0.05）;Compared with DEX+I/R group,MDA in ATI+DEX+I/R group was significantly increased（P<0.05）,and in YOH+DEX+I/R group was also significantly increased（P<0.01）.It is suggested that selectiveα₂-adrenergic receptor antagonist atipamezole or yohimbine block the locus coeruleus adrenergic system and antagonize the central anti-inflammatory effect of dexmedetomidine.Immunohistochemical results showed that the density of Iba-1 positive cells in hippocampal CA1 region was significantly different among all groups（P<0.01）.Compared with CON group and SHAM group,the density of Iba-1 positive cells in I/R group was significantly increased（P<0.01）.Compared with I/R group,the density of Iba-1 positive cells in hippocampus of DEX+I/R group,DSP-4+I/R group and DSP-4+DEX+I/R group was significantly decreased（P<0.01）.Both dexmedetomidine and DSP-4 pretreatment can reduce the accumulation of central microglia after intestinal ischemia/reperfusion injury.Conclusion:1.Intestinal ischemia/reperfusion injury can increase the expression of tyrosine hydroxylase positive neurons in locus coeruleus,promote the release of norepinephrine from hippocampus and aggravate the impairment of memory function after operation.2.Dexmetomidine,a selectiveα₂-adrenoceptor agonist,can reduce intestinal mucosal injury caused by ischemia/reperfusion,reduce chiu’s score and reduce the impairment of memory function after operation.3.α₂-adrenoceptor antagonists atipamezole and yohimbine could antagonize the cognitive protective effect of dexmetomidine,which proved thatα₂-adrenoceptor was involved in the protective mechanism of cognitive function.4.DSP-4 lesion of locus coeruleus adrenergic neurons could inhibit the release of norepinephrine,reduce the contents of IL-1β,TNF-αand MDA in the brain,and reduce the occurrence of postoperative memory impairment.It is suggested that locus ceruleus-norepinephrine system participates in central inflammatory response and regulates postoperative cognitive function.5.Intestinal ischemia/reperfusion injury can increase the contents of IL-1β,TNF-αand MDA in the brain and increase the density of microglia in the hippocampus,indicating that postoperative central nervous inflammation is related to the occurrence of postoperative cognitive dysfunction.6.Dexmedetomidine preconditioning can reduce the contents of IL-1β,TNF-αand MDA in the brain,reduce the density of Iba-1 positive microglia in the hippocampus,and reduce the impairment of memory function after operation,indicating that centralα₂-adrenoceptor agonist has the effect of anti-inflammation and alleviating neurological impairment.In conclusion,intestinal ischemia/reperfusion injury can cause not only systemic inflammatory response,but also central nervous system inflammation,significantly increase the expression of tyrosine hydroxylase in locus ceruleus adrenergic neurons and promote the release of central norepinephrine,resulting in postoperative cognitive impairment.Destruction of locus ceruleus adrenergic neurons can inhibit brain inflammation and reduce postoperative memory impairment.Centralα₂-adrenoceptor agonist could inhibit the activation of adrenergic neurons and improve the early postoperative cognitive impairment caused by intestinal ischemia/reperfusion injury by stimulatingα₂-adrenoceptor in locus ceruleus.On the contrary,α₂-adrenergic receptor antagonists atimazole and yohimbine could antagonize their cognitive protective effects.It is suggested that the excitation ofα₂-adrenoceptor in locus ceruleus can reduce the release of central norepinephrine,inhibit inflammation,prevent neurological damage and reduce the occurrence of postoperative cognitive dysfunction.更多还原