手术创伤对老年鼠术后认知功能损害和海马区炎性因子表达的研究
摘要
术后认知功能障碍(postoperative cognitive dysfunction, POCD)是指手术麻醉后数天内发生的意识、认知、定向、思维、记忆以及睡眠等方面的紊乱,是一种可逆的、具有波动性的急性精神紊乱综合征;通常又被称为术后精神障碍、术后谵妄等。POCD是脑功能暂时性机能障碍,可导致死亡率增加、康复延迟、并发症增多、住院天数延长和医疗费用增加等,严重时甚至影响病人出院后的生活质量。POCD常发生于老年人,最早发现于老年病人心脏手术后,近年来发现在非心脏手术病人也有较高的发生率,目前日益受到研究者的关注。由于诊断标准不同,观察病人例数不同,POCD发生率的报道也不一致。大量研究发现,POCD多发生于65岁以上的老年患者,发病率约为3%-61%,是年轻患者的2-10倍。Seymour等研究显示,年龄大于75岁的老年病人POCD的发生率比年龄在65-75岁的病人高3倍。Moller等通过对1218例非心脏手术老年病人36个月的调查发现,术后1周POCD的发生率与年龄增加、麻醉时间延长、受教育程度低、二次手术、术后感染和呼吸系统并发症等许多因素有关,但术后3个月POCD的发生率仅与年龄有关。
POCD确切的发病机制尚不明确。国外研究证实,POCD常常是多种因素协同作用的结果。易发因素包括高龄、高血压、糖尿病、长期服用某些药物、酗酒、感官缺陷、心理和环境因素等。促发因素包括应激反应、创伤、手术、术中出血和输血、脑血流降低、脑血管微栓子的形成、低血压、术后低氧血症、血压波动以及电解质紊乱等。患者存在的基础疾病如糖尿病、高血压、冠心病、脑梗死与POCD的发病关系密切。有学者认为,与老年性痴呆的发病机制一样,POCD可能是在神经系统老化的基础上,由麻醉、手术等外界因素诱发或加重的退行性改变,其发病机制是多种因素的综合作用。文献研究报道低血压和低氧血症并不影响POCD的发病率,因此更多的研究转向了手术创伤本身是否引起POCD.
既往研究认为全麻患者POCD的发生率要高于椎管内麻醉和局麻患者,近年研究表明,老年人POCD与麻醉方式间无明显相关性。Williams对全膝关节置换术患者随机实行全身麻醉和硬膜外麻醉,在术后1周和6个月POCD发生率无明显区别。美国20个医院的一项对年龄大于60岁髋关节骨折患者的回顾性研究发现,麻醉技术(全麻和局麻)对术后精神状态没有影响。
几项假说提出来解释POCD的可能机制,但现在还不清楚POCD的发生是否直接与手术创伤有关。已有文献证实创伤应激能激活外周的免疫系统,引起炎性因子的释放,导致认知功能的破坏。与年龄相关的炎性因子表达改变也有报道,包括炎性因子IL-1β, IL-6和TNF-α表达的增加。大量的研究证实,中枢系统炎性因子表达增加,尤其是在海马区可导致长时程增强效应(long term potentiation, LTP)的破坏和海马介导的认知功能的损伤。
手术创伤激活外周免疫系统引起炎症反应,释放各种炎性因子。免疫学研究发现组织损伤可激活巨噬细胞、单核细胞、成纤维细胞和内皮细胞。这些细胞的激活是对组织损伤最早期的分子反应,释放各种炎性介质,包括各种因子,补体和氧自由基,比如IL-1,IL-6和TNF。外周炎性因子通过直接激活转运系统或者间接通过刺激迷走神经两种途径透过血脑屏障。外周炎性因子进入和影响中枢神经系统的机制需要进一步的研究。在正常生理情况下,炎性因子在中枢神经系统不表达,在病理状态下表达迅速升高。外周的炎性因子通过直接或者间接途径作用于中枢神经系统。炎性因子可结合中枢神经系统相应的受体,激活小胶质细胞和血管内皮细胞,引起一系列的炎症反应和其它效应。外周的炎症反应与手术患者神经认知功能的下降有关。
手术创伤不仅激活外周的免疫反应,同时也激活中枢神经系统的免疫反应。外周的炎性因子、术前的微小栓子和应激反应都可以引起炎症反应。以往研究发现,炎性反应最重要的标志是小胶质细胞的激活。与巨噬细胞作用相似,小胶质细胞就如同一个清道夫,在受到刺激时会产生炎性因子。感染、缺血和炎症反应通过多种分子机制激活小胶质细胞。激活的小胶质细胞可释放可溶的炎性分子,包括炎性因子、类花生酸类物质、补体、兴奋性氨基酸、过氧化物和NO。Wan发现脾切除术引起胶质细胞激活导致海马区炎性反应,伴随着认知功能的下降。但至今还没有很好的方法解释手术创伤引起的炎性反应。Kalman发现冠脉搭桥的患者脑术后一周脊液炎性因子IL-6明显升高,术后6个月抗炎因子IL-4明显升高。这显示免疫调节反应参与了术后神经系统的损伤和心脏手术术后的并发症。炎性因子引起认知功能下降可通过影响神经递质的产生、影响突触可塑性和神经毒性发挥作用。因此手术创伤引起的中枢神经炎症反应或者外周炎症反应可引起POCD。本研究拟探讨手术创伤对老年鼠和成年鼠术后认知功能和海马区炎症因子表达的影响。
材料与方法
一、成年鼠和老年鼠手术创伤模型的建立
成年鼠和老年鼠分别随机分为对照组(每组12只)、麻醉组(每组36只)和手术组(每组36只)。所有实验动物都维持室温23℃,黑白周期12小时轮转,给与充足的食物和水。在实验结束后处死实验鼠后粗率检查老鼠有无明显疾病(脾测量法和有无肿瘤)。不健康的大鼠排除。所有动物都在这样的环境熟悉7-10天。对照组SD大鼠均不做特殊处理。麻醉组实验动物单纯接受1.5-2%异氟醚麻醉,然后行气管插管和机械通气。手术组SD大鼠行肝部分切除术。具体步骤：手术切口处备皮、消毒,在上腹部正中线做一小切口,游离切除左侧肝脏,伤口用0.25%布比卡因浸润,然后用缝线缝合,所有手术步骤均无菌操作。手术待老鼠恢复后放回笼子里各自饲养。麻醉组和手术组分别在术后1、3、7天利用反转的morris水迷宫进行行为学测试。试验结束后,测试完毕的老鼠1/2断头处死后立即取海马放在液氮中保存测量mRNA,剩余的1/2处死后经心脏灌注肝素化的生理盐水,然后在体灌注4%多聚甲醛,动物的脑组织被取出置于4%多聚甲醛中做免疫组化。
二、空间记忆能力-Morris水迷宫
一个直径1米水深30厘米的圆形水槽(水温24-26℃),一个圆形的透明平台置于水面下0.5厘米,水槽四周贴有如正方形、菱形等不同的图形,实验鼠要根据这些图形确定它的游行线路来找到水面下的平台。老鼠在手术前六天进行训练,每天三次,每次实验时,首先将实验鼠放在平台上30S,让其熟悉周围环境,然后让头部背对平台随机放入水槽的四个象限,自由游泳60S,或者找到平台,如果在60S内没找到平台,引导它找到平台,在平台上待30S,重复上述实验。在第7天实行肝部分切除术,术后第1、3、7天重复Morris水迷宫实验,不同的是将平台放在对面的象限,让实验鼠放在上面熟悉30s。这种倒转的水迷宫测验老鼠能否迅速学习和记忆平台的位置。置在水迷宫上方的摄像机连接计算机,记录游泳的速度、到达平台的时间和游行距离。
三、免疫组织化学
海马组织做5umm石蜡切片,载玻片上免疫染色。常规脱蜡至水,3%过氧化氢灭活内源性酶,热修复后,正常山羊血清封闭,神经胶质酸性蛋白(GFAP)、S100β和突触素分别用兔抗GFAP抗体(博士德)、小鼠抗S100β抗体(博士德)、兔抗突触素抗体(博士德)4℃过夜,切片在室温下用磷酸盐缓冲液冲洗三次。生物素化山羊抗兔IgG或者羊抗小鼠IgG 25℃孵育2h, SABC37℃孵育20 min,室温下DAB显色,镜下控制反应时间,以阳性产物呈棕黄色,背景淡棕色为度。苏木素复染,显微镜观察,照相。每只动物每隔5张取1张切片,共取5张利用图像分析管理系统进行图象分析统计处理。
四、RNA的提取和通过定量RT-PCR测量海马区细胞因子mRNA
RNA的提取：所有RNA都是用Trizol试剂从匀浆海马组织中分离提取。
cDNA的合成：cDNA的合成主要利用RNA PCR Kit (AMV) Ver.3.0试剂盒合成。
PCR扩增：2ul的cDNA在50ul的反应液里用2U的Taq多聚酶进行扩增。反应液包括5ul 10*PCR缓冲液,1.5mmMgCl2 buffer, dNTPs(各0.2 mM),以及0.4mmIL-1β、IL-6和TNF-1α的基因特异性引物。PCR扩增。在PCR扩增目的基因时,加入一对内参照β-actin的特异性引物,同时扩增内参DNA,作为对照。5ul的扩增产物进行琼脂糖凝胶电泳。条带密度采用凝胶图像分析系统定量。mRNA的量由特异性基因的]mRNA和β-actin(一种看家基因)mRNA的比值来表示。
五、统计学分析
RT-PCR和免疫组化所得的数据实行两因素方差分析,年龄(成年、老年)和干预因素(对照、手术)作为因变量。术前Morris水迷宫每次计算所得到数据使用重复测量方差分析。术后Morris水迷宫计算所得到数据实行两因素方差分析。若方差分析揭示结果有统计学意义,则使用post hoc Student's t检验。所得数据以x±s表示,P<0.05认为有统计学意义。
结果
一、老年鼠和成年鼠游行的速度、到达平台的时间和游行距离
利用重复测量方差分析六天的训练数据显示,时间(p<0.001)和年龄(p<0.001)在到达平台的时间和游行的距离上有统计学差异(图1A&1B),在游行速度上没有统计学差异(图1C)。这些结果证明随着训练时间的增多,老年鼠和成年鼠都能改善空间记忆和学习能力,但老年鼠需游行更长的时间和更远的距离才能到达平台。游行速度上无统计学差异说明老年鼠游行时间和距离的增加并不是因为运动能力下降造成的。利用翻转的morris水迷宫检测实验鼠手术后的空间记忆和学习能力。分析到达平台的时间和游行的距离显示年龄(p<0.001),手术(p<0.001)和年龄×手术(p=0.047,p=0.054,)均有统计学差异(图2A&2B),游行速度在年龄和手术之间没有差异(图2C)。成年鼠的空间记忆学习能力(到达平台的时间和游行的距离)在术后第1天(p<0.001)受到破坏,在术后第3天恢复(p=0.848,p=0.973,)。老年鼠的空间记忆学习能力(到达平台的时间和游行的距离)在术后第1天(p<0.001)受到破坏,并且持续到术后第3天(p=0.002,p=0.001),在术后第7天恢复。单纯的麻醉药物并没有明显破坏实验鼠术后的认知功能。这些数据说明手术创伤破坏了实验鼠的空间记忆和学习能力,并且高龄易化了这种破坏,老年鼠认知功能破坏表现的更强烈,持续时间更长。
二、手术创伤对海马区炎性因子表达的的影响
利用两因素方差分析海马区炎性因子mRNA的表达变化,年龄(p<0.001),手术(p<0.001)和年龄×手术(p<0.001, p<0.001 and p=0.003)在IL-1 p,IL-6和TNF-α上有统计学差异(图3A-3C),成年鼠和老年鼠海马区IL-1p,IL-6和TNF-α在基础水平存在统计学差异(p=0.041,p=0.012和p=0.035)。单纯麻醉因素没有明显改变成年鼠和老年鼠海马区炎性因子的表达。成年鼠海马区IL-1p和IL-6仅在手术后第1天(p<0.001)明显增高,但老年鼠海马区炎性因子增多持续到手术后3天(p<0.001,).(图3A&3B)。TNF-αmRNA仅在老年鼠手术后1天(p<0.001)明显增加,成年鼠在任何时间点都没有明显改变(p=0.095)(图3C)。这些结果证明手术创伤可明显增加海马区炎性因子的表达,这种改变尤其在老年鼠明显。
三、手术创伤对海马区GFAP和S100p蛋白表达的影响
本实验探讨成年鼠和老年鼠在手术创伤后炎性因子表达差异的原因,我们利用免疫组化探讨了海马区GFAP和S100β蛋白表达的变化。两因素方差分析显示年龄(p<0.001)、手术(p<0.001)和年龄×手术(p<0.001)对GFAP和S100β表达有统计学差异,GFAP和S100β在成年鼠和老年鼠在基础水平也有统计学差异(图4A&4B)。当增加的GFAP阳性细胞在成年鼠术后第3天(p=0.09)恢复后,老年鼠GFAP阳性细胞在术后第3天(p<0.001)仍然明显增加,在术后第7天(p=0.823)恢复(图4A)。S100p有与GFAP同样的变化趋势(图4B)。
四、手术创伤对海马区CD200和CD200R mRNA表达的影响
为了探讨手术创伤激活海马区胶质细胞的潜在机制,我们测定了海马区CD200和CD200R mRNA的表达变化。CD200在成年鼠和老年鼠术后第一天表达下调(p<0.001),术后第三天恢复正常(p=0.067,p=0.582)(图5A)。在任何时间点,手术创伤并没有明显改变成年鼠和老年鼠CD200R mRNA的表达水平(图5B)。这些结果证明手术创伤引起的胶质细胞激活部分机制可能是因为激活了海马区CD200 mRNA的表达。
结论
1、手术创伤而不是麻醉因素导致了术后认知功能障碍,高龄加剧了认知功能的破坏。
2、手术创伤导致了海马区炎性因子IL-1p,IL-6和TNF-a的表达,老年鼠炎性因子表达更加剧烈,持续时间更长。炎性因子的表达同术后认知功能改变平行,可能介导了术后认知功能障碍。
3、手术创伤引起海马区胶质细胞的激活,老年鼠比成年鼠激活更加明显,持续时间更长。
4、手术创伤导致海马区CD200表达下降,外周手术创伤引起的海马区胶质细胞的激活部分机制是由于CD200表达下调。
Introduction
Elderly patients undergoing surgical intervention often suffer from postoperative cognitive dysfunction (POCD), a condition characterized by the progressive deterioration of cognitive function, following surgery. These cognitive impairments, either transient or permanent, affect a significant number of patients, and result in prolonged hospitalization and delayed recovery from illness. POCD is more frequently reported following cardiac surgery and is also known to occur in other surgical procedures.
Although the neurobiological basis of POCD remains unknown, major risk factors, such as advanced age, poor education, preexisting cognitive impairment, severity of coexisting illness, duration of anesthesia, respiratory complications and second operation, have been identified. Of these, age has been increasingly reported as the most prominent risk factor for the development of POCD. Surprisingly, hypoxaemia or hypotensive episodes do not appear to influence the incidence of POCD,suggesting more involvement of surgical trauma itself in the impairment of cognitive function.
Several theories have been advanced to explain the mechanisms of POCD. It remains unclear whether its occurrence is directly related to surgical trauma. Stress activates the peripheral innate immune system, resulting in the release of inflammatory mediators, which impairs cognitive function [13]. Age-related neuroinflammatory changes have been reported, including the increased expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. A substantial body of evidence indicates that significantly elevated expression of pro-inflammatory cytokines, particularly in the hippocampus, results in impairments in long-term potentiation (LTP) and performance deficits in hippocampal-mediated cognitive tests. In the present study, we investigated the effects of partial hepatectomy on spatial learning and memory and hippocampal pro-inflammatory cytokines in adult and aged rats.
Materials and methods
Animals
Both adult (3-6 months old) and aged (20-24 months old) Sprague-Dawley male rats were randomly divided into a total of six groups:control (n=12 for each group), anesthesia alone (n=36 for each group), and operation group (n=36 for each group). All animals were housed in a temperature controlled room on a 12-h light and 12-h dark cycle with ad libitum access to food and water. All rats were adapted to their environment for a minimum of 7-10 days before experimentation. Control animals received sterile saline to control for effects of injection stress. The anesthesia alone group received 1.5% to 2% isoflurane with intubation and mechanical ventilation. The operation group underwent partial hepatectomy under general anesthesia. In brief, the liver was exposed through a 1-2 cm midline abdominal incision. The left lateral lobes of the liver (approximately corresponding to 30% of the organ) were excised. The wound was then infiltrated with 0.25% bupivacaine, and closed by sterile suture. All experimental procedures were performed in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Experimental procedures
Rats were sacrificed on postoperative days 1,3, and 7 (n=12/time point) after spatial working tests. Hippocampal tissues of half of the rats in each group were quickly dissected and stored at-70℃until they were assayed for central cytokine mRNAs. The remaining animals in each group were killed and perfused transcardially and postfixed for immunohistochemistry.
Cognitive testing
The Morris water maze (MWM) is a hippocampal-dependent test of spatial learning for rodents. Rats were placed on the platform for 30 s before the start of each trial, and released into the water facing the wall of the pool from one of four randomly assigned release points (N, W, S and E). In all trials, rats were allowed to swim until they landed on the platform. If a rat failed to find the platform within 60 s, it was usually picked up and placed on the platform for 15 s. The animal remained on the platform for 30 s between trials. Rats were trained with the platform in a fixed location for three trials per day for 6 consecutive days. Animals underwent surgery on day 7. On postoperative days 1,3,7 rats were subjected to a reversal test in which the platform was relocated to the opposite quadrant of the pool. Reversal learning reveals whether animals can extinguish their initial learning of the platform's position and acquire a direct path to the new goal position. Swimming distance, speed, and latency to the platform were recorded by video tracking mounted on the ceiling and digital images were analyzed by water maze software (HVS image, United Kingdom).
Brain cytokine mRNA extraction and reverse-transcription PCR
Total RNA was extracted from homogenization of 200 mg hippocampal tissue samples per manufacturer's instructions for TRIZOL Reagent (Invitrogen, US). Reverse-transcription polymerase chain reacrtion (RT-PCR) was performed as previously reported. Briefly, synthesis of the first strand of complementary DNA was conducted using RNA PCR Kit (AMV) Version 3.0 (TaKaRa, Japan). Reverse transcription was performed at 30℃for 10 min, 42℃for 30 min and terminated by heating to 99℃for 5 min. The products were amplified by PCR. Primer sequences and amplification sizes are given in Table 1. The amount of mRNA was expressed as a ratio of densitometric measurements derived from target mRNA andβ-actin.
Immunohistochemistry
Immunohistochemistry was performed as previously reported. Sections were deparaffinized and rehydrated, then incubated with 3% H2O2 in methanol at room temperature for 10 min to block endogenous peroxidase and washed three times with PBS, and blocked in 1% normal goat serum at room temperature for 30 min. Primary antibodies were used as follows:mouse rabbit anti-rat glial fibrillary acidic protein (GFAP) antibody(1:80; Sigma) and mouse anti-rat S100βantibody (1:200; Sigma). Incubation with primary antibody was carried out at 4℃for overnight, followed by rinsing several times. Biotinylated goat anti-rabbit or goat anti-mouse IgG secondary antibody was applied at 37℃for 30 min. After thorough washing, the reaction product was visualized using the DAB method. The sections were counterstained with hematoxylin, dehydrated, and mounted. Control samples were run in parallel omitting primary antibody. Every fifth section was selected and generated 5-10 sections per reference space in a systematic-random manner. The positive cells in the hippocampus were estimated using the MetaMorph software system (US).
Statistical Analysis
Data from immunohistochemistry and RT-PCR were analyzed with two-way ANOVA in which age and operation were dependent variables. Repeated-measures ANOVA as a two-way design was used to analyze training behavioral parameters. A separate two-way ANOVA examined the effects of age and operation on working memory performance during reversal testing. Post hoc Student's t-test was employed when ANOVA revealed significance. A p-value< 0.05 was considered to be statistically significant.
Results
Aged rats exhibit impaired performance on spatial learning and memory exacerbated by surgical trauma
To evaluate the effects of surgery on hippocampus-dependent spatial learning and memory, a MWM was constructed. Repeated-measures of ANOVA of swim data revealed significant effects of day (p<0.001) and age (p<0.001) on both latency and distance (Figs. 1A & 1B), but not on speed (p=0.194) (Fig. 1C).These results indicated that while both adult and aged rats showed improvement in spatial learning and memory over time, aged rats swam further and longer in order to reach the target quadrant during the acquisition phase. The lack of effect of speed suggests that the poorer performance of aged rats did not result from lack of motivation or reduced motor ability. Analysis of distance and latency to platform during reversal testing revealed significant effects of age (p<0.001), operation (p<0.001) and age×operation interaction (p=0.047, p=0.054, respectively) (Figs. 2A & 2B), but no main effect of speed (p=0.634) (Fig.2C). Impaired performance on distance and latency parameters was found on postoperative day 1 (p< 0.001) in adult rats, and improved on day 3 (p=0.848, p=0.973, respectively). In aged rats, the same impairments were found on postoperative day 1 (p< 0.001) and continued to day 3 (p=0.002, p=0.001, respectively), only improving on day 7 (p=0.966, p=0.775, respectively) (Figs. 2A & 2B). Anesthesia alone did not significantly impair cognitive function on days 1,3,7 postoperatively (data not shown). These results demonstrate that the surgical procedure impaired spatial learning and memory, and induced a prolonged sickness response in aged rats.
Aged rats exhibit elevated pro-inflammatory cytokine expression exacerbated by surgical trauma
To investigate whether anesthesia or surgical trauma alters pro-inflammatory cytokine expression, hippocampal IL-1(3, IL-6 and TNF-a mRNA were measured. Two-way ANOVA of hippocampal IL-1β, IL-6 and TNF-a expression revealed significant effects of age (p< 0.001), operation (p< 0.001) and age×operation interaction (p< 0.001, p< 0.001 and p= 0.003, respectively) (Figs.3A-3C). Significant differences were observed for basal hippocampal IL-1β, IL-6 and TNF-a mRNA levels between adult and aged rats (p=0.041, p=0.012, and p=0.035 respectively). Anesthesia alone did not significantly alter hippocampal pro-inflammatory cytokine levels in adult or aged animals when compared to their age-matched naive controls at any time point (data not shown). IL-1βand IL-6 levels were upregulated by surgical trauma on postoperative day 1 (p<0.001) in adult rats; these cytokines remained upregulated until postoperative day 3 in aged rates (p< 0.001). (Figs.3A & 3B). TNF-a mRNA only increased on day 1 postoperatively in aged subjects (p< 0.001), but did not do so in adult subjects (p= 0.095) (Fig.3C). There was an amplified and prolonged pro-inflammatory cytokine response in aged brains following surgical procedure. These results indicate that the surgical procedure performed resulted in a further amplification of pro-inflammatory cytokine levels in aged hippocampal tissues.
Aged rats exhibit upregulated GFAP and S100βexpression in the hippocampus following surgical trauma
To explore why there were age-related differences in cytokine expression following surgery procedure, hippocampal GFAP and S100βwere examined. Analysis of GFAP and S100βrevealed significant effects of age (p<0.001, respectively) and operation (p<0.001, respectively), and agexoperation interaction (p<0.001, respectively) (Figs.4A & 4B). Significant differences were observed for basal hippocampal GFAP and S100βbetween adult and aged rats (p=0.039, p=0.002, respectively). While the GFAP-positive cells of the adult subjects downregulated on day 3 (p=0.09), GFAP expression in aged subjects remained significantly upregulated on day 3 (p<0.001) and improved on day 7 postoperatively (p=0.823) (Fig.4A). A similar pattern change was seen with S100β(Fig. 4B). These results indicate that aging may potentiate hippocampal glial cell activation when challenged by the surgical procedure performed here.
CD200 and CD200R mRNA expression following surgical trauma
To investigate potential mechanisms on how surgical trauma sensitizes glial cell activation, CD200 and CD200R mRNA were measured. CD200 expression was downregulated on postoperative day 1 in adult and aged rats (p< 0.001, respectively), and improved on day 3 (p=0.067, p=0.582, respectively) (Fig.5A). Surgical trauma did not significantly alter hippocampal CD200R levels in adult and aged animals at any time point (data not show) (Fig. 5B). These results indicate that trauma-induced sensitization of glial cell activation may be mediated, in part by the downregulation of hippocampal CD200 expression.
Discussion
The findings of this study indicate that moderate surgical trauma induced an exaggerated neuroinflammatory response and exacerbated cognitive function impairments in aged rats when compared to their adult counterparts. Anesthesia alone did not impair cognitive function or alter hippocampal pro-inflammatory cytokine levels in adult and aged rats. Our study revealed an age-related increase in the vulnerability to cognitive impairments triggered by surgical trauma. Postoperative cognitive impairment appears to parallel an increase in central pro-inflammatory cytokines expression.
Cytokines have been described as double-edged swords. They protect, repair but also impair neuronal function during excessive or chronic neuroinflammation.It has been postulated that overexpression of central inflammatory cytokines may lead to profound disturbances in sensory-motor coordination and cognition. The hippocampus, a brain region which highly expresses pro-inflammatory cytokine receptors, appears to be more sensitive to excessive or prolonged cytokine exposure.
Blockade of pro-inflammatory cytokine receptors in the brain blunts many aspects of the sickness response to peripheral immune challenge. It is known that the central administration of exogenous IL-1βinduces pro-inflammatory immune responses and cognitive deficits which are attenuated by intracerebroventricular administration of IL-1 receptor antagonist. Other studies have indicated that administration of IL-6 neutralizing antibodies prolongs LTP and improves spatial alternation behavior and facilitates recovery from LPS-induced sickness behavior. These studies emphasize the role of pro-inflammatory cytokines in regulating behavioral and cognitive changes.
The differential inflammatory responses in the hippocampus following surgical trauma challenge between aged and adult subjects may be explained by age-specific differences in glial cells. Glial cells generally exhibit a quiescent phenotype in the healthy adult brain. In normal aging however, glial cells are primed. In this primed state, the cells exhibit increases in the expression of GFAP, S100βand major histocompatibility complex-Ⅱ. Primed glial cells do not secrete appreciable levels of pro-inflammatory cytokines under basal conditions, but they are hyper-responsive to secondary stimuli and can produce an exaggerated and prolonged neuroinflammatory response when further provoked.Thus, the inflammatory response in aged brain is magnified when challenged with peripheral stimulus such as surgical trauma, resulting in greater cognitive impairments.
Peripheral immune challenge is transmitted to the brain via multiple humoral and neural routes. This immune-to-brain signaling results in the de novo production of pro-inflammatory cytokines within the brain, largely by glial cell. Vagal sensory pathways are known to be important in mediating cytokine-induced sickness behavior. In rats, subdiaphragmatic vagotomy attenuates the upregulation of IL-1βin the hippocampus and alleviates behavioral depression induced by peripheral injection of LPS. Moreover, vagal stimulation also increases IL-1βin the brain. These findings explain why activation of the peripheral innate immune system by surgical trauma induces brain glial cell activation to produce pro-inflammatory cytokines responsible for cognitive deficits.
To investigate potential mechanisms on how surgical trauma sensitizes glial cell activation, CD200 and CD200R were measured. CD200 expressed on the surface of neurons is thought to constitutively maintain glial cell in a quiescent state through interactions with its receptor CD200R. Recent evidence indicates that CD200 expression is reduced in aged animals, suggesting that reduced neuronal control of glial cell may be an age-related feature which results in glial cell activation. The effects of surgical trauma on glial cell in aged rats suggest that trauma-induced sensitization of glial cell may be mediated, in part, by the attenuation of neuronal control of microglia through downregulation of hippocampal CD200 expression. Therefore, modulating the inhibitory CD200-CD200R interplay to silence glial cell activation may be a promising strategy for improving recovery from sickness and reducing neurobehavioral deficits.
In conclusion, the results of this study indicate that pro-inflammatory cytokines may play a significant role in disrupting normal cognitive function in the hippocampus. Surgical trauma resulted in exaggerated and prolonged neuroinflammatory response in the hippocampus and greater cognitive impairments in aged rats when compared to those of their adult counterparts. Inhibition of neuroinflammation triggered by surgical trauma may be a promising strategy for POCD prevention.
Acknowledgements
This research was financed by a grant from the Natural Science Foundation of Liaoning Education Committee (Grant No.2008796).
POCD确切的发病机制尚不明确。国外研究证实,POCD常常是多种因素协同作用的结果。易发因素包括高龄、高血压、糖尿病、长期服用某些药物、酗酒、感官缺陷、心理和环境因素等。促发因素包括应激反应、创伤、手术、术中出血和输血、脑血流降低、脑血管微栓子的形成、低血压、术后低氧血症、血压波动以及电解质紊乱等。患者存在的基础疾病如糖尿病、高血压、冠心病、脑梗死与POCD的发病关系密切。有学者认为,与老年性痴呆的发病机制一样,POCD可能是在神经系统老化的基础上,由麻醉、手术等外界因素诱发或加重的退行性改变,其发病机制是多种因素的综合作用。文献研究报道低血压和低氧血症并不影响POCD的发病率,因此更多的研究转向了手术创伤本身是否引起POCD.
既往研究认为全麻患者POCD的发生率要高于椎管内麻醉和局麻患者,近年研究表明,老年人POCD与麻醉方式间无明显相关性。Williams对全膝关节置换术患者随机实行全身麻醉和硬膜外麻醉,在术后1周和6个月POCD发生率无明显区别。美国20个医院的一项对年龄大于60岁髋关节骨折患者的回顾性研究发现,麻醉技术(全麻和局麻)对术后精神状态没有影响。
几项假说提出来解释POCD的可能机制,但现在还不清楚POCD的发生是否直接与手术创伤有关。已有文献证实创伤应激能激活外周的免疫系统,引起炎性因子的释放,导致认知功能的破坏。与年龄相关的炎性因子表达改变也有报道,包括炎性因子IL-1β, IL-6和TNF-α表达的增加。大量的研究证实,中枢系统炎性因子表达增加,尤其是在海马区可导致长时程增强效应(long term potentiation, LTP)的破坏和海马介导的认知功能的损伤。
手术创伤激活外周免疫系统引起炎症反应,释放各种炎性因子。免疫学研究发现组织损伤可激活巨噬细胞、单核细胞、成纤维细胞和内皮细胞。这些细胞的激活是对组织损伤最早期的分子反应,释放各种炎性介质,包括各种因子,补体和氧自由基,比如IL-1,IL-6和TNF。外周炎性因子通过直接激活转运系统或者间接通过刺激迷走神经两种途径透过血脑屏障。外周炎性因子进入和影响中枢神经系统的机制需要进一步的研究。在正常生理情况下,炎性因子在中枢神经系统不表达,在病理状态下表达迅速升高。外周的炎性因子通过直接或者间接途径作用于中枢神经系统。炎性因子可结合中枢神经系统相应的受体,激活小胶质细胞和血管内皮细胞,引起一系列的炎症反应和其它效应。外周的炎症反应与手术患者神经认知功能的下降有关。
手术创伤不仅激活外周的免疫反应,同时也激活中枢神经系统的免疫反应。外周的炎性因子、术前的微小栓子和应激反应都可以引起炎症反应。以往研究发现,炎性反应最重要的标志是小胶质细胞的激活。与巨噬细胞作用相似,小胶质细胞就如同一个清道夫,在受到刺激时会产生炎性因子。感染、缺血和炎症反应通过多种分子机制激活小胶质细胞。激活的小胶质细胞可释放可溶的炎性分子,包括炎性因子、类花生酸类物质、补体、兴奋性氨基酸、过氧化物和NO。Wan发现脾切除术引起胶质细胞激活导致海马区炎性反应,伴随着认知功能的下降。但至今还没有很好的方法解释手术创伤引起的炎性反应。Kalman发现冠脉搭桥的患者脑术后一周脊液炎性因子IL-6明显升高,术后6个月抗炎因子IL-4明显升高。这显示免疫调节反应参与了术后神经系统的损伤和心脏手术术后的并发症。炎性因子引起认知功能下降可通过影响神经递质的产生、影响突触可塑性和神经毒性发挥作用。因此手术创伤引起的中枢神经炎症反应或者外周炎症反应可引起POCD。本研究拟探讨手术创伤对老年鼠和成年鼠术后认知功能和海马区炎症因子表达的影响。
材料与方法
一、成年鼠和老年鼠手术创伤模型的建立
成年鼠和老年鼠分别随机分为对照组(每组12只)、麻醉组(每组36只)和手术组(每组36只)。所有实验动物都维持室温23℃,黑白周期12小时轮转,给与充足的食物和水。在实验结束后处死实验鼠后粗率检查老鼠有无明显疾病(脾测量法和有无肿瘤)。不健康的大鼠排除。所有动物都在这样的环境熟悉7-10天。对照组SD大鼠均不做特殊处理。麻醉组实验动物单纯接受1.5-2%异氟醚麻醉,然后行气管插管和机械通气。手术组SD大鼠行肝部分切除术。具体步骤：手术切口处备皮、消毒,在上腹部正中线做一小切口,游离切除左侧肝脏,伤口用0.25%布比卡因浸润,然后用缝线缝合,所有手术步骤均无菌操作。手术待老鼠恢复后放回笼子里各自饲养。麻醉组和手术组分别在术后1、3、7天利用反转的morris水迷宫进行行为学测试。试验结束后,测试完毕的老鼠1/2断头处死后立即取海马放在液氮中保存测量mRNA,剩余的1/2处死后经心脏灌注肝素化的生理盐水,然后在体灌注4%多聚甲醛,动物的脑组织被取出置于4%多聚甲醛中做免疫组化。
二、空间记忆能力-Morris水迷宫
一个直径1米水深30厘米的圆形水槽(水温24-26℃),一个圆形的透明平台置于水面下0.5厘米,水槽四周贴有如正方形、菱形等不同的图形,实验鼠要根据这些图形确定它的游行线路来找到水面下的平台。老鼠在手术前六天进行训练,每天三次,每次实验时,首先将实验鼠放在平台上30S,让其熟悉周围环境,然后让头部背对平台随机放入水槽的四个象限,自由游泳60S,或者找到平台,如果在60S内没找到平台,引导它找到平台,在平台上待30S,重复上述实验。在第7天实行肝部分切除术,术后第1、3、7天重复Morris水迷宫实验,不同的是将平台放在对面的象限,让实验鼠放在上面熟悉30s。这种倒转的水迷宫测验老鼠能否迅速学习和记忆平台的位置。置在水迷宫上方的摄像机连接计算机,记录游泳的速度、到达平台的时间和游行距离。
三、免疫组织化学
海马组织做5umm石蜡切片,载玻片上免疫染色。常规脱蜡至水,3%过氧化氢灭活内源性酶,热修复后,正常山羊血清封闭,神经胶质酸性蛋白(GFAP)、S100β和突触素分别用兔抗GFAP抗体(博士德)、小鼠抗S100β抗体(博士德)、兔抗突触素抗体(博士德)4℃过夜,切片在室温下用磷酸盐缓冲液冲洗三次。生物素化山羊抗兔IgG或者羊抗小鼠IgG 25℃孵育2h, SABC37℃孵育20 min,室温下DAB显色,镜下控制反应时间,以阳性产物呈棕黄色,背景淡棕色为度。苏木素复染,显微镜观察,照相。每只动物每隔5张取1张切片,共取5张利用图像分析管理系统进行图象分析统计处理。
四、RNA的提取和通过定量RT-PCR测量海马区细胞因子mRNA
RNA的提取：所有RNA都是用Trizol试剂从匀浆海马组织中分离提取。
cDNA的合成：cDNA的合成主要利用RNA PCR Kit (AMV) Ver.3.0试剂盒合成。
PCR扩增：2ul的cDNA在50ul的反应液里用2U的Taq多聚酶进行扩增。反应液包括5ul 10*PCR缓冲液,1.5mmMgCl2 buffer, dNTPs(各0.2 mM),以及0.4mmIL-1β、IL-6和TNF-1α的基因特异性引物。PCR扩增。在PCR扩增目的基因时,加入一对内参照β-actin的特异性引物,同时扩增内参DNA,作为对照。5ul的扩增产物进行琼脂糖凝胶电泳。条带密度采用凝胶图像分析系统定量。mRNA的量由特异性基因的]mRNA和β-actin(一种看家基因)mRNA的比值来表示。
五、统计学分析
RT-PCR和免疫组化所得的数据实行两因素方差分析,年龄(成年、老年)和干预因素(对照、手术)作为因变量。术前Morris水迷宫每次计算所得到数据使用重复测量方差分析。术后Morris水迷宫计算所得到数据实行两因素方差分析。若方差分析揭示结果有统计学意义,则使用post hoc Student's t检验。所得数据以x±s表示,P<0.05认为有统计学意义。
结果
一、老年鼠和成年鼠游行的速度、到达平台的时间和游行距离
利用重复测量方差分析六天的训练数据显示,时间(p<0.001)和年龄(p<0.001)在到达平台的时间和游行的距离上有统计学差异(图1A&1B),在游行速度上没有统计学差异(图1C)。这些结果证明随着训练时间的增多,老年鼠和成年鼠都能改善空间记忆和学习能力,但老年鼠需游行更长的时间和更远的距离才能到达平台。游行速度上无统计学差异说明老年鼠游行时间和距离的增加并不是因为运动能力下降造成的。利用翻转的morris水迷宫检测实验鼠手术后的空间记忆和学习能力。分析到达平台的时间和游行的距离显示年龄(p<0.001),手术(p<0.001)和年龄×手术(p=0.047,p=0.054,)均有统计学差异(图2A&2B),游行速度在年龄和手术之间没有差异(图2C)。成年鼠的空间记忆学习能力(到达平台的时间和游行的距离)在术后第1天(p<0.001)受到破坏,在术后第3天恢复(p=0.848,p=0.973,)。老年鼠的空间记忆学习能力(到达平台的时间和游行的距离)在术后第1天(p<0.001)受到破坏,并且持续到术后第3天(p=0.002,p=0.001),在术后第7天恢复。单纯的麻醉药物并没有明显破坏实验鼠术后的认知功能。这些数据说明手术创伤破坏了实验鼠的空间记忆和学习能力,并且高龄易化了这种破坏,老年鼠认知功能破坏表现的更强烈,持续时间更长。
二、手术创伤对海马区炎性因子表达的的影响
利用两因素方差分析海马区炎性因子mRNA的表达变化,年龄(p<0.001),手术(p<0.001)和年龄×手术(p<0.001, p<0.001 and p=0.003)在IL-1 p,IL-6和TNF-α上有统计学差异(图3A-3C),成年鼠和老年鼠海马区IL-1p,IL-6和TNF-α在基础水平存在统计学差异(p=0.041,p=0.012和p=0.035)。单纯麻醉因素没有明显改变成年鼠和老年鼠海马区炎性因子的表达。成年鼠海马区IL-1p和IL-6仅在手术后第1天(p<0.001)明显增高,但老年鼠海马区炎性因子增多持续到手术后3天(p<0.001,).(图3A&3B)。TNF-αmRNA仅在老年鼠手术后1天(p<0.001)明显增加,成年鼠在任何时间点都没有明显改变(p=0.095)(图3C)。这些结果证明手术创伤可明显增加海马区炎性因子的表达,这种改变尤其在老年鼠明显。
三、手术创伤对海马区GFAP和S100p蛋白表达的影响
本实验探讨成年鼠和老年鼠在手术创伤后炎性因子表达差异的原因,我们利用免疫组化探讨了海马区GFAP和S100β蛋白表达的变化。两因素方差分析显示年龄(p<0.001)、手术(p<0.001)和年龄×手术(p<0.001)对GFAP和S100β表达有统计学差异,GFAP和S100β在成年鼠和老年鼠在基础水平也有统计学差异(图4A&4B)。当增加的GFAP阳性细胞在成年鼠术后第3天(p=0.09)恢复后,老年鼠GFAP阳性细胞在术后第3天(p<0.001)仍然明显增加,在术后第7天(p=0.823)恢复(图4A)。S100p有与GFAP同样的变化趋势(图4B)。
四、手术创伤对海马区CD200和CD200R mRNA表达的影响
为了探讨手术创伤激活海马区胶质细胞的潜在机制,我们测定了海马区CD200和CD200R mRNA的表达变化。CD200在成年鼠和老年鼠术后第一天表达下调(p<0.001),术后第三天恢复正常(p=0.067,p=0.582)(图5A)。在任何时间点,手术创伤并没有明显改变成年鼠和老年鼠CD200R mRNA的表达水平(图5B)。这些结果证明手术创伤引起的胶质细胞激活部分机制可能是因为激活了海马区CD200 mRNA的表达。
结论
1、手术创伤而不是麻醉因素导致了术后认知功能障碍,高龄加剧了认知功能的破坏。
2、手术创伤导致了海马区炎性因子IL-1p,IL-6和TNF-a的表达,老年鼠炎性因子表达更加剧烈,持续时间更长。炎性因子的表达同术后认知功能改变平行,可能介导了术后认知功能障碍。
3、手术创伤引起海马区胶质细胞的激活,老年鼠比成年鼠激活更加明显,持续时间更长。
4、手术创伤导致海马区CD200表达下降,外周手术创伤引起的海马区胶质细胞的激活部分机制是由于CD200表达下调。
Introduction
Elderly patients undergoing surgical intervention often suffer from postoperative cognitive dysfunction (POCD), a condition characterized by the progressive deterioration of cognitive function, following surgery. These cognitive impairments, either transient or permanent, affect a significant number of patients, and result in prolonged hospitalization and delayed recovery from illness. POCD is more frequently reported following cardiac surgery and is also known to occur in other surgical procedures.
Although the neurobiological basis of POCD remains unknown, major risk factors, such as advanced age, poor education, preexisting cognitive impairment, severity of coexisting illness, duration of anesthesia, respiratory complications and second operation, have been identified. Of these, age has been increasingly reported as the most prominent risk factor for the development of POCD. Surprisingly, hypoxaemia or hypotensive episodes do not appear to influence the incidence of POCD,suggesting more involvement of surgical trauma itself in the impairment of cognitive function.
Several theories have been advanced to explain the mechanisms of POCD. It remains unclear whether its occurrence is directly related to surgical trauma. Stress activates the peripheral innate immune system, resulting in the release of inflammatory mediators, which impairs cognitive function [13]. Age-related neuroinflammatory changes have been reported, including the increased expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. A substantial body of evidence indicates that significantly elevated expression of pro-inflammatory cytokines, particularly in the hippocampus, results in impairments in long-term potentiation (LTP) and performance deficits in hippocampal-mediated cognitive tests. In the present study, we investigated the effects of partial hepatectomy on spatial learning and memory and hippocampal pro-inflammatory cytokines in adult and aged rats.
Materials and methods
Animals
Both adult (3-6 months old) and aged (20-24 months old) Sprague-Dawley male rats were randomly divided into a total of six groups:control (n=12 for each group), anesthesia alone (n=36 for each group), and operation group (n=36 for each group). All animals were housed in a temperature controlled room on a 12-h light and 12-h dark cycle with ad libitum access to food and water. All rats were adapted to their environment for a minimum of 7-10 days before experimentation. Control animals received sterile saline to control for effects of injection stress. The anesthesia alone group received 1.5% to 2% isoflurane with intubation and mechanical ventilation. The operation group underwent partial hepatectomy under general anesthesia. In brief, the liver was exposed through a 1-2 cm midline abdominal incision. The left lateral lobes of the liver (approximately corresponding to 30% of the organ) were excised. The wound was then infiltrated with 0.25% bupivacaine, and closed by sterile suture. All experimental procedures were performed in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Experimental procedures
Rats were sacrificed on postoperative days 1,3, and 7 (n=12/time point) after spatial working tests. Hippocampal tissues of half of the rats in each group were quickly dissected and stored at-70℃until they were assayed for central cytokine mRNAs. The remaining animals in each group were killed and perfused transcardially and postfixed for immunohistochemistry.
Cognitive testing
The Morris water maze (MWM) is a hippocampal-dependent test of spatial learning for rodents. Rats were placed on the platform for 30 s before the start of each trial, and released into the water facing the wall of the pool from one of four randomly assigned release points (N, W, S and E). In all trials, rats were allowed to swim until they landed on the platform. If a rat failed to find the platform within 60 s, it was usually picked up and placed on the platform for 15 s. The animal remained on the platform for 30 s between trials. Rats were trained with the platform in a fixed location for three trials per day for 6 consecutive days. Animals underwent surgery on day 7. On postoperative days 1,3,7 rats were subjected to a reversal test in which the platform was relocated to the opposite quadrant of the pool. Reversal learning reveals whether animals can extinguish their initial learning of the platform's position and acquire a direct path to the new goal position. Swimming distance, speed, and latency to the platform were recorded by video tracking mounted on the ceiling and digital images were analyzed by water maze software (HVS image, United Kingdom).
Brain cytokine mRNA extraction and reverse-transcription PCR
Total RNA was extracted from homogenization of 200 mg hippocampal tissue samples per manufacturer's instructions for TRIZOL Reagent (Invitrogen, US). Reverse-transcription polymerase chain reacrtion (RT-PCR) was performed as previously reported. Briefly, synthesis of the first strand of complementary DNA was conducted using RNA PCR Kit (AMV) Version 3.0 (TaKaRa, Japan). Reverse transcription was performed at 30℃for 10 min, 42℃for 30 min and terminated by heating to 99℃for 5 min. The products were amplified by PCR. Primer sequences and amplification sizes are given in Table 1. The amount of mRNA was expressed as a ratio of densitometric measurements derived from target mRNA andβ-actin.
Immunohistochemistry
Immunohistochemistry was performed as previously reported. Sections were deparaffinized and rehydrated, then incubated with 3% H2O2 in methanol at room temperature for 10 min to block endogenous peroxidase and washed three times with PBS, and blocked in 1% normal goat serum at room temperature for 30 min. Primary antibodies were used as follows:mouse rabbit anti-rat glial fibrillary acidic protein (GFAP) antibody(1:80; Sigma) and mouse anti-rat S100βantibody (1:200; Sigma). Incubation with primary antibody was carried out at 4℃for overnight, followed by rinsing several times. Biotinylated goat anti-rabbit or goat anti-mouse IgG secondary antibody was applied at 37℃for 30 min. After thorough washing, the reaction product was visualized using the DAB method. The sections were counterstained with hematoxylin, dehydrated, and mounted. Control samples were run in parallel omitting primary antibody. Every fifth section was selected and generated 5-10 sections per reference space in a systematic-random manner. The positive cells in the hippocampus were estimated using the MetaMorph software system (US).
Statistical Analysis
Data from immunohistochemistry and RT-PCR were analyzed with two-way ANOVA in which age and operation were dependent variables. Repeated-measures ANOVA as a two-way design was used to analyze training behavioral parameters. A separate two-way ANOVA examined the effects of age and operation on working memory performance during reversal testing. Post hoc Student's t-test was employed when ANOVA revealed significance. A p-value< 0.05 was considered to be statistically significant.
Results
Aged rats exhibit impaired performance on spatial learning and memory exacerbated by surgical trauma
To evaluate the effects of surgery on hippocampus-dependent spatial learning and memory, a MWM was constructed. Repeated-measures of ANOVA of swim data revealed significant effects of day (p<0.001) and age (p<0.001) on both latency and distance (Figs. 1A & 1B), but not on speed (p=0.194) (Fig. 1C).These results indicated that while both adult and aged rats showed improvement in spatial learning and memory over time, aged rats swam further and longer in order to reach the target quadrant during the acquisition phase. The lack of effect of speed suggests that the poorer performance of aged rats did not result from lack of motivation or reduced motor ability. Analysis of distance and latency to platform during reversal testing revealed significant effects of age (p<0.001), operation (p<0.001) and age×operation interaction (p=0.047, p=0.054, respectively) (Figs. 2A & 2B), but no main effect of speed (p=0.634) (Fig.2C). Impaired performance on distance and latency parameters was found on postoperative day 1 (p< 0.001) in adult rats, and improved on day 3 (p=0.848, p=0.973, respectively). In aged rats, the same impairments were found on postoperative day 1 (p< 0.001) and continued to day 3 (p=0.002, p=0.001, respectively), only improving on day 7 (p=0.966, p=0.775, respectively) (Figs. 2A & 2B). Anesthesia alone did not significantly impair cognitive function on days 1,3,7 postoperatively (data not shown). These results demonstrate that the surgical procedure impaired spatial learning and memory, and induced a prolonged sickness response in aged rats.
Aged rats exhibit elevated pro-inflammatory cytokine expression exacerbated by surgical trauma
To investigate whether anesthesia or surgical trauma alters pro-inflammatory cytokine expression, hippocampal IL-1(3, IL-6 and TNF-a mRNA were measured. Two-way ANOVA of hippocampal IL-1β, IL-6 and TNF-a expression revealed significant effects of age (p< 0.001), operation (p< 0.001) and age×operation interaction (p< 0.001, p< 0.001 and p= 0.003, respectively) (Figs.3A-3C). Significant differences were observed for basal hippocampal IL-1β, IL-6 and TNF-a mRNA levels between adult and aged rats (p=0.041, p=0.012, and p=0.035 respectively). Anesthesia alone did not significantly alter hippocampal pro-inflammatory cytokine levels in adult or aged animals when compared to their age-matched naive controls at any time point (data not shown). IL-1βand IL-6 levels were upregulated by surgical trauma on postoperative day 1 (p<0.001) in adult rats; these cytokines remained upregulated until postoperative day 3 in aged rates (p< 0.001). (Figs.3A & 3B). TNF-a mRNA only increased on day 1 postoperatively in aged subjects (p< 0.001), but did not do so in adult subjects (p= 0.095) (Fig.3C). There was an amplified and prolonged pro-inflammatory cytokine response in aged brains following surgical procedure. These results indicate that the surgical procedure performed resulted in a further amplification of pro-inflammatory cytokine levels in aged hippocampal tissues.
Aged rats exhibit upregulated GFAP and S100βexpression in the hippocampus following surgical trauma
To explore why there were age-related differences in cytokine expression following surgery procedure, hippocampal GFAP and S100βwere examined. Analysis of GFAP and S100βrevealed significant effects of age (p<0.001, respectively) and operation (p<0.001, respectively), and agexoperation interaction (p<0.001, respectively) (Figs.4A & 4B). Significant differences were observed for basal hippocampal GFAP and S100βbetween adult and aged rats (p=0.039, p=0.002, respectively). While the GFAP-positive cells of the adult subjects downregulated on day 3 (p=0.09), GFAP expression in aged subjects remained significantly upregulated on day 3 (p<0.001) and improved on day 7 postoperatively (p=0.823) (Fig.4A). A similar pattern change was seen with S100β(Fig. 4B). These results indicate that aging may potentiate hippocampal glial cell activation when challenged by the surgical procedure performed here.
CD200 and CD200R mRNA expression following surgical trauma
To investigate potential mechanisms on how surgical trauma sensitizes glial cell activation, CD200 and CD200R mRNA were measured. CD200 expression was downregulated on postoperative day 1 in adult and aged rats (p< 0.001, respectively), and improved on day 3 (p=0.067, p=0.582, respectively) (Fig.5A). Surgical trauma did not significantly alter hippocampal CD200R levels in adult and aged animals at any time point (data not show) (Fig. 5B). These results indicate that trauma-induced sensitization of glial cell activation may be mediated, in part by the downregulation of hippocampal CD200 expression.
Discussion
The findings of this study indicate that moderate surgical trauma induced an exaggerated neuroinflammatory response and exacerbated cognitive function impairments in aged rats when compared to their adult counterparts. Anesthesia alone did not impair cognitive function or alter hippocampal pro-inflammatory cytokine levels in adult and aged rats. Our study revealed an age-related increase in the vulnerability to cognitive impairments triggered by surgical trauma. Postoperative cognitive impairment appears to parallel an increase in central pro-inflammatory cytokines expression.
Cytokines have been described as double-edged swords. They protect, repair but also impair neuronal function during excessive or chronic neuroinflammation.It has been postulated that overexpression of central inflammatory cytokines may lead to profound disturbances in sensory-motor coordination and cognition. The hippocampus, a brain region which highly expresses pro-inflammatory cytokine receptors, appears to be more sensitive to excessive or prolonged cytokine exposure.
Blockade of pro-inflammatory cytokine receptors in the brain blunts many aspects of the sickness response to peripheral immune challenge. It is known that the central administration of exogenous IL-1βinduces pro-inflammatory immune responses and cognitive deficits which are attenuated by intracerebroventricular administration of IL-1 receptor antagonist. Other studies have indicated that administration of IL-6 neutralizing antibodies prolongs LTP and improves spatial alternation behavior and facilitates recovery from LPS-induced sickness behavior. These studies emphasize the role of pro-inflammatory cytokines in regulating behavioral and cognitive changes.
The differential inflammatory responses in the hippocampus following surgical trauma challenge between aged and adult subjects may be explained by age-specific differences in glial cells. Glial cells generally exhibit a quiescent phenotype in the healthy adult brain. In normal aging however, glial cells are primed. In this primed state, the cells exhibit increases in the expression of GFAP, S100βand major histocompatibility complex-Ⅱ. Primed glial cells do not secrete appreciable levels of pro-inflammatory cytokines under basal conditions, but they are hyper-responsive to secondary stimuli and can produce an exaggerated and prolonged neuroinflammatory response when further provoked.Thus, the inflammatory response in aged brain is magnified when challenged with peripheral stimulus such as surgical trauma, resulting in greater cognitive impairments.
Peripheral immune challenge is transmitted to the brain via multiple humoral and neural routes. This immune-to-brain signaling results in the de novo production of pro-inflammatory cytokines within the brain, largely by glial cell. Vagal sensory pathways are known to be important in mediating cytokine-induced sickness behavior. In rats, subdiaphragmatic vagotomy attenuates the upregulation of IL-1βin the hippocampus and alleviates behavioral depression induced by peripheral injection of LPS. Moreover, vagal stimulation also increases IL-1βin the brain. These findings explain why activation of the peripheral innate immune system by surgical trauma induces brain glial cell activation to produce pro-inflammatory cytokines responsible for cognitive deficits.
To investigate potential mechanisms on how surgical trauma sensitizes glial cell activation, CD200 and CD200R were measured. CD200 expressed on the surface of neurons is thought to constitutively maintain glial cell in a quiescent state through interactions with its receptor CD200R. Recent evidence indicates that CD200 expression is reduced in aged animals, suggesting that reduced neuronal control of glial cell may be an age-related feature which results in glial cell activation. The effects of surgical trauma on glial cell in aged rats suggest that trauma-induced sensitization of glial cell may be mediated, in part, by the attenuation of neuronal control of microglia through downregulation of hippocampal CD200 expression. Therefore, modulating the inhibitory CD200-CD200R interplay to silence glial cell activation may be a promising strategy for improving recovery from sickness and reducing neurobehavioral deficits.
In conclusion, the results of this study indicate that pro-inflammatory cytokines may play a significant role in disrupting normal cognitive function in the hippocampus. Surgical trauma resulted in exaggerated and prolonged neuroinflammatory response in the hippocampus and greater cognitive impairments in aged rats when compared to those of their adult counterparts. Inhibition of neuroinflammation triggered by surgical trauma may be a promising strategy for POCD prevention.
Acknowledgements
This research was financed by a grant from the Natural Science Foundation of Liaoning Education Committee (Grant No.2008796).
引文
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46张挺杰,皋源,江燕等.老年病人术后精神障碍的发生率和病因分析.临床麻醉学杂志.2003；19(2)：98-100.
47周静,周苏明.老年人手术后精神障碍临床分析.实用老年医学.2006,2(1)：46-48.
48 Rasmussen L S. Postoperative cognitive dysfunction:incidence and prevention. Best Pract Res Clin Anaesthesiol.2006,20(2):315-330.
49 Muller SV, Krause N, SchmidtM, et al. Cognitive dysfunction after abdominal surgery in elderly patients. Z Gerontol Geriatr.2004,37:475-485.
50 Zmavar V,et al.Assessment of neurocognitive impairment after off-pump and on-pump techniques for coronary artery bypass graft surgery:prospective randomized controlled trial.BMJ.2002:1268-1273.
51 Newman S, Stygall J, Hirani S, Shaefi S, Maze M. Postoperative cognitive dysfunction after noncardiac surgery:a systematic review. Anesthesiology.2007; 106:572-590.
52 Maze M, Cibelli M, Grocott HP. Taking the lead in research into postoperative cognitive dysfunction. Anesthesiology.2008; 108:1-2.
53 Thomson LM, Sutherland RJ. Systemic administration of lipopolysaccharide and interleukin-lbeta have different effects on memory consolidation. Brain Res Bull.2005; 67: 24-29.
54 Wilson CJ, Finch CE, Cohen HJ. Cytokines and cognition—the case for a head-totoe inflammatory paradigm. J Am Geriatr Soc.2002; 50:2041-2056.
55 Chen J, Buchanan JB, Sparkman NL, Godbout JP, Freund GG, Johnson RW. Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain Behav Immun.2008; 22:301-311.
56 Godbout JP, Chen J, Abraham J, et al. Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. FASEB J.2005; 19: 1329-1331.
57 Kurosawa S, Kato M. Anesthetics, immune cells, and immune responses. J Anesth.2008; 22: 263-277.
58 Ni Choileain N, Redmond HP. Cell response to surgery. Arch Surg.2006; 141:1132-1140.
59 Ramlawi B, Rudolph JL, Mieno S, et al. C-Reactive protein and inflammatory response associated to neurocognitive decline following cardiac surgery.Surgery.2006; 140:221-226.
60 Kalman J, Juhasz A, Bogats G, et al. Elevated levels of inflammatory biomarkers in the cerebrospinal fluid after coronary artery bypass surgery are predictors of cognitive decline. Neurochem Int.2006; 48:177-180.
61 Rojo LE, Fernandez JA, Maccioni AA, Jimenez JM, Maccioni RB. Neuroinflammation: implications for the pathogenesis and molecular diagnosis of Alzheimer's disease. Arch Med Res.2008; 39:1-16.
62 Cauli O, Rodrigo R, Piedrafita B, Boix J, Felipo V. Inflammation and hepatic encephalopathy: ibuprofen restores learning ability in rats with portacaval shunts. Hepatology.2007; 46: 514-519.
63 Tobinick EL, Gross H. Rapid cognitive improvement in Alzheimer's disease following perispinal etanercept administration. J Neuroinflamm.2008; 5:2-6.
64 odbout JP, Johnson RW. Age and neuroinflammation:a lifetime of psychoneuroimmune consequences, Immunol Allergy Clin North Am.2009; 29:321-337.
65 asmussen LS. Postoperative cognitive dysfunction:incidence and prevention, Best Pract Res Clin Anaesthesiol.2006; 20:315-330.
66 Barbut D, Yao FS, Hager DN, Kavanaugh P, Trifiletti RR, Gold JP. Comparison of transcranial Doppler ultrasonography and transesophageal echocardiography to monitor emboli during coronary artery bypass surgery, Stroke.1996;27:87-90.
67 Liu YH, Wang DX, Li LH, Wu XM, Shan GJ, Su Y, Li J, Yu QJ, Shi CX, Huang YN, Sun W. The effects of cardiopulmonary bypass on the number of cerebral microemboli and the incidence of cognitive dysfunction after coronary artery bypass graft surgery, Anesth Analg.2009; 109: 1013-1022.
68 Bekker AY, Weeks EJ. Cognitive function after anesthesia in the elderly, Best Pract Res Clin Anaesthesiol.2003; 17:259-272.
69 Litaker D, Locala J, Franco K, Bronson DL, Tannous Z. Preoperative risk factors for postoperative delirium, Gen Hosp Psychiatry.2001; 23:84-89.
70 Ancelin ML, Roquefeuil GD, Ledesert B, Bonnel F, Cheminal JC, Ritchie K. Exposure to anesthetic agents, cognitive functioning and depressive symptomatology in the elderly, Br J Psychiatry.2001;178:360-366.
71 Ramaiah R, Lam AM. Postoperative cognitive dysfunction in the elderly. Anesthesiol Clin.2009; 27:485-496.
72 Buchanan JB, Sparkman NL, Chen J, Johnson RW. Cognitive and neuroinflammatory consequences of mild repeated stress are exacerbated in aged mice. Psychoneuroendocrinology.2008;33:755-765.
73 Morgan CJ, Mofeez A, Brandner B et al. Acute effects of ketamine onmemory systems and p sychotic symp toms in healthy volunteers. Neurop sychopharmacology.2004; 29(1):208-212.
74 Honey RA, Turner DC, Honey GD et al. Subdissociative dose ketamine p roduces a deficit in manipulation but notmaintenance of the contents ofworkingmemory. Neurop sychopharmacology.2003; 28 (11):2037-2041.
75 Pfenninger EG, DurieuxME, Himmelseher S. Cognitive impairment after small-does ketamine isomers in comparison to equianalgesic racemic ketamine in human volunteers. Anesthesiology.2002; 96 (2):357-362.
76 Curran HV, Morgan C. Cognitive, dissociative and p sychotogenic effects of ketamine in recreational userson the night of drug use and 3 days later. Addiction.2000; 95(4):575-581.
77 Culley DJ, BaxterM, Yukhananov R et al. The memory effects of general anesthesia persist forweeks in young and aged rats. Anesth Analg.2003; 96 (4):1004-1007.
78 Rasmussen L S. Postoperative cognitive dysfunction:incidence and prevention. Best Pract Res Clin Anaesthesiol.2006,20(2):315-330.
79 Muller SV, Krause N, SchmidtM, et al. Cognitive dysfunction after abdominal surgery in elderly patients. Z Gerontol Geriatr.2004,37:475-485.
80 H.A. Rosczyk,N.L. Sparkman,et al. Neuroinflammation and cognitive function in aged mice following minor surgery. Experimental Gerontology.2008; 43:840-846.
81 Vorhees CV, Williams MT. Morris water maze:procedures for assessing spatial and related forms of learning and memory, Nat Protoc.2006; 1:848-858.
82 Griffin WS, Liu L, Li Y, Mrak R, Barger S. Interleukin-1 mediates Alzheimer and Lewy body pathologies, J Neuroinflammation.2006; 3:5-14.
83 Parnet P, Kelley KW, Bluthe RM, Dantzer R. Expression and regulation of interleukin-1 receptors in the brain:role in cytokines-induced sickness behavior, J Neuroimmunol.2002; 125: 5-14.
84 Sheeran P, Hall GM. Cytokines in anaesthesia. Br J Anaesth 1997;78:201-219.
85 Wilson CJ, Finch CE, Cohen HJ. Cytokines and cognition-the case for a head to-toe inflammatory paradigm. J Am Geriatr Soc.2002; 50:2041-2056.
86 Minagar A, Shapshak P, Fujimura R, Ownby R, Heyes M, Eisdorfer C. The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIV-associated dementia, Alzheimer disease, and multiple sclerosis. J Neurol Sci.2002; 202: 13-23.
87 Kalman J, Juhasz A, Bogats G, et al. Elevated levels of inflammatory biomarkers in the cerebrospinal fluid after coronary artery bypass surgery are predictors of cognitive decline. Neurochem Int.2006; 48:177-180.
88 Johnson JD, O'Connor KA, Watkins LR, Maier SF. The role of IL-lbeta in stress-induced sensitization of pro-inflammatory cytokine and corticosterone responses, Neuroscience 2004;127,569-577.
89 Balschun D, Wetzel W, Del Rey A, Pitossi F, Schneider H, Zuschratter W, Besedovsky HO. Interleukin-6:a cytokine to forget, FASEB J 2004; 18,1788-1790.
90 Bluthe RM, Michaud B, Poli V, Dantzer R. Role of IL-6 in cytokine induced sickness behavior:a study IL-6 deficient mice, Physiol Behav.2000; 70:367-373.
91 Ramaiah R, Lam AM.Postoperative cognitive dysfunction in the elderly. Anesthesiol Clin. 2009; 27(3):485-496.
92 Folstein MF, Folstein SE, Mc Hugh P. "Mini-mental state". A practicalmethod for grading the cognitive state of patients for the clinician. J Psychiatr Res.1999; 12 (3):189-194.
93 Rasmussen LS, Johnson T, Kuipers HM et al. Does anaesthesia cause postoperative cognitive dysfunction? A randomised study of regional versus general anaesthesia in 438 elderly patients. Acta Anaesthesiol Scand.2003; 47 (3):260-265
94 Combrinck MI, Perry VH, Cunningham C. Peripheral infection evokes exaggerated sickness behavior in pre-clinical murine prion disease, Neuroscience.2002; 112:7-11.
95 Konsman JP, Parnet P, Dantzer R. Cytokine-induced sickness behaviour:mechanisms and implications, Trends Neurosci.2002; 25:154-159.
96 Konsman JP, Luheshi GN, Bluthe RM, Dantzer R. The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune signals; a functional anatomical analysis, Eur J Neurosci.2000; 12:4434-4446.
97 Feldman SA. A comparative study of four premedications.Anaest-hesia,2003; 18:169-184.
98 Maier SF. Bi-directional immune-brain communication:Implications for understanding stress, pain, and cognition, Brain Behav Immun.2003; 17:69-85.
99 Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, Song Y, Jenmalm M, Gorman D, McClanahan T, Liu MR, Brown MH, Sedgwick JD, Phillips JH, Barclay AN. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200, J Immunol.2003; 171:3034-3046.
1 Selnes O A, McKhann G M.Neurocognitive complications after coronary artery bypass surgery. Ann Neurol.2010; 57(5):615-621.
2 Saravay SM, Kap LM, Kurek J et al. How do delirium and dementia increase length of stay of eldery generalmedical inpatients. Psychosomatics.2004; 45 (3):235-239.
3 Rasmussen LS, Steentoft A, Rasmussen H et al. Benzodiazep ines and postoperative cognitive dysfunction in the elderly. Br J Anaesth.2009; 83 (4):585-592.
4 Moller JT, Cluitmans P, Rasmussen LS et al. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet.1998; 351(9):857-861.
5 Marcantonio ER, Goldman L, Mangione CM, et al. A clinical prediction rule for delirium after elective noncardiacsurgery. JAMA.1994;271:134-139.
6 Bekker AY, Weeks EJ. Cognitive function after anaesthesia in the elderly. Best Pract Res Clin Anaesthesiol.2010; 17:259-272.
7 Crichnik K P,Ijsselmniden A J,et al.Cognitive decline after major noncardiac operations:a prelimingary prospective study.Ann Thorac Surg.1999,68(5):1786-1791.
8 Ramaiah R, Lam AM.Postoperative cognitive dysfunction in the elderly. Anesthesiol Clin. 2009; 27(3):485-496.
9 Folstein MF, Folstein SE, Mc Hugh P. "Mini-mental state". A practicalmethod for grading the cognitive state of patients for the clinician. J Psychiatr Res.2010; 12 (3):189-193.
10 Pratico C, D. Quattrone, T. Lucanto, A. Amato, O. Penna, et al. Drugs of anesthesia acting on central cholinergic system may cause post-operative cognitive dysfunction and delirium.Medical Hypothesis.2005; 65:972-982.
11 Rasmussen LS, Johnson T, Kuipers HM et al. Does anaesthesia cause postoperative cognitive dysfunction? A randomised study of regional versus general anaesthesia in 438 elderly patients. Acta Anaesthesiol Scand.2003; 47 (3):260-268.
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13 Feldman SA. A comparative study of four premedications.Anaest-hesia.2009; 18:169-184
14 Dodds C, Allison J. Postoperative cognitive deficit in t he elderly surgical patient. Br J Anaesth.1998;81:449-462.
15 Morgan CJ, Mofeez A, Brandner B et al. Acute effects of ketamine onmemory systems and p sychotic symp toms in healthy volunteers. Neurop sychopharmacology.2004; 29(1):208-212.
16 Honey RA, Turner DC, Honey GD et al. Subdissociative dose ketamine p roduces a deficit in manipulation but notmaintenance of the contents ofworkingmemory. Neurop sychopharmacology.2003; 28 (11):2037-2042.
17 Pfenninger EG, DurieuxME, Himmelseher S. Cognitive impairment after small-does ketamine isomers in comparison to equianalgesic racemic ketamine in human volunteers. Anesthesiology.2009; 96 (2):357-362.
18 Curran HV, Morgan C. Cognitive, dissociative and p sychotogenic effects of ketamine in recreational userson the night of drug use and 3 days later. Addiction.2000; 95(4):575-583.
19 Culley DJ, BaxterM, Yukhananov R et al. The memory effects of general anesthesia persist forweeks in young and aged rats. Anesth Analg.2003; 96 (4):1004-1009.
20 Rasmussen L S. Postoperative cognitive dysfunction:incidence and prevention. Best Pract Res Clin Anaesthesiol.2006; 20(2):315-330.
21 Muller SV, Krause N, SchmidtM, et al. Cognitive dysfunction after abdominal surgery in elderly patients. Z Gerontol Geriatr.2004; 37:475-485.
22 Zmavar V,et al.Assessment of neurocognitive impairment after off-pump and on-pump techniques for coronary artery bypass graft surgery:prospective randomized controlled trial.BMJ.2002; 7375:1268-1272.
23 Canet J, Raeder J, Rasmussen LS et al. Cognitive dysfunction afterminor surgery in the elderly. Acta Anaesthesiol Scand.2003; 47 (10):1204-1208.
24 Byrick RJ, Kay JC, Mazer CD et al. Dynamic characteristics of cerebral lip idmicroemboli: videomicroscopy studies in rats. Anesth Analg.2010,97 (6):1789-1793.
25 张挺杰,皋源,江燕等.老年病人术后精神障碍的发生率和病因分析.临床麻醉学杂志.2003： 19 (2) ：98-101.
26周静,周苏明.老年人手术后精神障碍临床分析.实用老年医学.2006;2(1)：46-51.
27 Linstedt U,Meyer O,et al. Serum concentration of S-100 protein in assessment of cognitive dysfunction after general anesthesia in different types of surgery [J].Acta Anaesthesiol Scand.2002; 46(4):384-389.
28 Yanjie Wan, M.D., Jing Xu, M.D,et al. Postoperative Impairment of Cognitive Function in Rats:A Possible Role for Cytokine-mediated Inflammation in the Hippocampus. Anesthesiology.2007; 106:436-443.
29 H.A. Rosczyk,N.L. Sparkman,et al. Neuroinflammation and cognitive function in aged mice following minor surgery. Experimental Gerontology.2008; 43:840-846.
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32 32 黄文,朱佩芳,王正国,等.老年人载脂蛋白E基因型与认知功能关系的研究.中国行为医学科学.2002;11(6)：614-617.
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35 Jevtovic T V, Hartman R E, et al. Early exposureto common anesthetic agents causeswidesp read neurodegeneration in the develop ing rat brain and persisten learning deficits.J Neurosci. 2003; 23 (3):876-882.
36王东信,吴新民,李军.利多卡因对心脏手术后病人早期认知功能障碍发生率的影响.中华麻醉学杂志.2004,24(2)：85-88.
37 L instedtU, MeyerO, Berkau A et al. Does intraoperative hyperventilation imp rove neurological functions of older patients after general anaesthesia? Anaesthesist.2002; 51(6): 457-461.
38朱科明,邓小明,马宇.异丙酚对ICU老年患者急性术后精神障碍的治疗.中国急救医学.2004：24(1)：15-18.
39马宇,朱科明,邓小明.老年患者术后发生急性瞻妄的临床分析.临床麻醉学杂志.2004；20：481-483.
2 Rohan D, Buggy DJ, Crowley S, Ling FK, Gallagher H, Regan C, Moriarty DC. Increased incidence of postoperative cognitive dysfunction 24 hr after minor surgery in the elderly, Can J Anaesth.2005; 52:137-142.
3 Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Canet J, Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T, Lauven PM, Kristensen PA, Biedler A, van Beem H, Fraidakis O, Silverstein JH, Beneken JE, Gravenstein JS. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study, ISPOCD investigators, International Study of Post-Operative Cognitive Dysfunction, Lancet.1998; 351:857-861.
4 Hudetz JA, Iqbal Z, Gandhi SD, Patterson KM, Hyde TF, Reddy DM, Hudetz AG, Warltier DC. Postoperative cognitive dysfunction in older patients with a history of alcohol abuse, Anesthesiology.2007; 106:423-430.
5 Bekker AY, Weeks EJ. Cognitive function after anesthesia in the elderly, Best Pract Res Clin Anaesthesiol.2003; 17:259-272.
6 Litaker D, Locala J, Franco K, Bronson DL, Tannous Z. Preoperative risk factors for postoperative delirium, Gen Hosp Psychiatry.2001; 23:84-89,.
7 Ancelin ML, Roquefeuil GD, Ledesert B, Bonnel F, Cheminal JC, Ritchie K. Exposure to anesthetic agents, cognitive functioning and depressive symptomatology in the elderly, Br J Psychiatry.2001;178:360-366.
8 Ramaiah R, Lam AM. Postoperative cognitive dysfunction in the elderly. Anesthesiol Clin. 2009;27:485-496.
9 Rasmussen LS. Postoperative cognitive dysfunction:incidence and prevention, Best Pract Res Clin Anaesthesiol.2006;20:315-330.
10 Barbut D, Yao FS, Hager DN, Kavanaugh P, Trifiletti RR, Gold JP. Comparison of transcranial Doppler ultrasonography and transesophageal echocardiography to monitor emboli during coronary artery bypass surgery, Stroke.1996;27:87-90.
11 Liu YH, Wang DX, Li LH, Wu XM, Shan GJ, Su Y, Li J, Yu QJ, Shi CX, Huang YN, Sun W. The effects of cardiopulmonary bypass on the number of cerebral microemboli and the incidence of cognitive dysfunction after coronary artery bypass graft surgery, Anesth Analg.2009; 109: 1013-1022.
12 Wan Y, Xu J, Ma D, Zeng Y, Cibelli M, Maze M. Postoperative impairment of cognitive function in rats:a possible role for cytokine-mediated inflammation in the hippocampus, Anesthesiology.2007;106:436-443.
13 uchanan JB, Sparkman NL, Chen J, Johnson RW. Cognitive and neuroinflammatory consequences of mild repeated stress are exacerbated in aged mice. Psychoneuroendocrinology. 2008;33:755-765.
14 odbout JP, Johnson RW. Age and neuroinflammation:a lifetime of psychoneuroimmune consequences, Immunol Allergy Clin North Am.2009;29:321-337.
15 Pickering M, Cumiskey D, O'Connor JJ. Actions of TNF-alpha on glutamatergic synaptic transmission in the central nervous system, Exp Physiol.2005;90:663-670.
16 Perry VH, Cunningham C, Holmes C. Systemic infections and inflammation affect chronic neurodegeneration, Nat Rev Immunol.2007;7:161-167.
17 Tanaka S, Ide M, Shibutani T, Ohtaki H, Numazawa S, Shioda S, Yoshida T. Lipopolysaccharide-induced microglial activation induces learning and memory deficits without neuronal cell death in rats, J Neurosci Res.2006;83:557-566.
18 Sparkman NL, Kohman RA, Scott VJ, Boehm GW. Bacterial endotoxin-induced behavioral alterations in two variations of the Morris water maze, Physiol Behav 2005; 86:244-251,.
19 Vorhees CV, Williams MT. Morris water maze:procedures for assessing spatial and related forms of learning and memory, Nat Protoc.2006; 1:848-858.
20 Griffin WS, Liu L, Li Y, Mrak R, Barger S. Interleukin-1 mediates Alzheimer and Lewy body pathologies, J Neuroinflammation.2006;3:5-14.
21 Barrientos R M, Higgins EA, Biedenkapp JC, Sprunger DB, Wright-Hardesty KJ, Watkins LR, Rudy JW, Maier SF. Peripheral infection and aging interact to impair hippocampal memory consolidation, Neurobiol Aging.2006; 27:723-732.
22 Parnet P, Kelley KW, Bluthe RM, Dantzer R. Expression and regulation of interleukin-1 receptors in the brain:role in cytokines-induced sickness behavior, J Neuroimmunol.2002; 125: 5-14.
23 Johnson JD, O'Connor KA, Watkins LR, Maier SF. The role of IL-lbeta in stress-induced sensitization of pro-inflammatory cytokine and corticosterone responses, Neuroscience.2004; 127:569-577.
24 Balschun D, Wetzel W, Del Rey A, Pitossi F, Schneider H, Zuschratter W, Besedovsky HO. Interleukin-6:a cytokine to forget, FASEB J.2004;18:1788-1790.
25 Bluthe RM, Michaud B, Poli V, Dantzer R. Role of IL-6 in cytokine induced sickness behavior:a study IL-6 deficient mice, Physiol Behav.2000; 70:367-373.
26 Godbout JP, Johnson RW. Age and neuroinflammation:a lifetime of psychoneuroimmune consequences, Neurol Clin.2006; 24:521-538.
27 Combrinck MI, Perry VH, Cunningham C. Peripheral infection evokes exaggerated sickness behavior in pre-clinical murine prion disease, Neuroscience.2002; 112:7-11.
28 Konsman JP, Parnet P, Dantzer R. Cytokine-induced sickness behaviour:mechanisms and implications, Trends Neurosci.2002; 25:154-159.
29 Konsman JP, Luheshi GN, Bluthe RM, Dantzer R. The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune signals; a functional anatomical analysis, Eur J Neurosci.2000; 12:4434-4446.
30 Maier SF. Bi-directional immune-brain communication:Implications for understanding stress, pain, and cognition, Brain Behav Immun.2003; 17:69-85.
31 Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, Song Y, Jenmalm M, Gorman D, McClanahan T, Liu MR, Brown MH, Sedgwick JD, Phillips JH, Barclay AN. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200, J Immunol.2003; 171:3034-3046.
32 M.G Frank, R.M. Barrientos, J.C. Biedenkapp, J.W. Rudy, L.R. Watkins, S.F. Maier, mRNA up-regulation of MHC II and pivotal pro-inflammatory genes in normal brain aging, Neurobiol Aging.2006; 27:717-722.
33 Selnes O A, McKhann G M.Neurocognitive complications after coronary artery bypass surgery [J]. Ann Neurol.2005; 57(5):615-621.
34 Saravay SM, Kap LM, Kurek J et al. How do delirium and dementia increase length of stay of eldery generalmedical inpatients. Psychosomatics.2004; 45 (3):235-241.
35 Rasmussen LS, Steentoft A, Rasmussen H et al. Benzodiazep ines and postoperative cognitive dysfunction in the elderly. Br J Anaesth.2009; 83 (4):585-589.
36 Moller JT, Cluitmans P, Rasmussen LS et al. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet.1998; 351(9):857-861.
37 Crichnik K P,Ijsselmniden A J,et al.Cognitive decline after major noncardiac operations:a prelimingary prospective study [J].Ann Thorac Surg.1999,68(5):1786-1791.
38 Marcantonio ER, Goldman L, Mangione CM, et al. A clinical prediction rule for delirium after elective noncardiacsurgery. JAMA.1994; 271:134-139.
39 Rohm KD, Piper SN, Suttner S, Schuler S, Boldt J. Early recovery, cognitive function and costs of a desflurane inhalational vs a total intravenous anaesthesia regimen in long-term surgery.. Acta Anaesthesiol Scand.2006; 50:14-18.
40 Farag E, Chelune GJ, Schubert A, Mascha EJ. Is depth of anesthesia, as assessed by the bispectral index, related to postoperative cognitive dysfunction and recovery. Anesth Analg. 2006;103:633-640.
41 Rasmussen LS, Johnson T, Kuipers HM, et al. Does anaesthesia cause postoperative cognitive dysfunction? A randomised study of regional versus general anaesthesia in 438 elderly patients. Acta Anaesthesiol Scand.2003; 47:260-266.
42 Dodds C, Allison J. Postoperative cognitive deficit in the elderly surgical patient. Br J Anaesth. 1998; 81 (3):449-457.
43 Folstein MF, Folstein SE, Mc Hugh P. "Mini-mental state". A practicalmethod for grading the cognitive state of patients for the clinician. J Psychiatr Res.1999; 12 (3):189-193.
44 Canet J, Raeder J, Rasmussen LS et al. Cognitive dysfunction afterminor surgery in the elderly. Acta Anaesthesiol Scand.2003,47 (10):1204-1208.
45 Byrick RJ, Kay JC, Mazer CD et al. Dynamic characteristics of cerebral lip idmicroemboli: videomicroscopy studies in rats. Anesth Analg.2003,97 (6):1789-1796.
46张挺杰,皋源,江燕等.老年病人术后精神障碍的发生率和病因分析.临床麻醉学杂志.2003；19(2)：98-100.
47周静,周苏明.老年人手术后精神障碍临床分析.实用老年医学.2006,2(1)：46-48.
48 Rasmussen L S. Postoperative cognitive dysfunction:incidence and prevention. Best Pract Res Clin Anaesthesiol.2006,20(2):315-330.
49 Muller SV, Krause N, SchmidtM, et al. Cognitive dysfunction after abdominal surgery in elderly patients. Z Gerontol Geriatr.2004,37:475-485.
50 Zmavar V,et al.Assessment of neurocognitive impairment after off-pump and on-pump techniques for coronary artery bypass graft surgery:prospective randomized controlled trial.BMJ.2002:1268-1273.
51 Newman S, Stygall J, Hirani S, Shaefi S, Maze M. Postoperative cognitive dysfunction after noncardiac surgery:a systematic review. Anesthesiology.2007; 106:572-590.
52 Maze M, Cibelli M, Grocott HP. Taking the lead in research into postoperative cognitive dysfunction. Anesthesiology.2008; 108:1-2.
53 Thomson LM, Sutherland RJ. Systemic administration of lipopolysaccharide and interleukin-lbeta have different effects on memory consolidation. Brain Res Bull.2005; 67: 24-29.
54 Wilson CJ, Finch CE, Cohen HJ. Cytokines and cognition—the case for a head-totoe inflammatory paradigm. J Am Geriatr Soc.2002; 50:2041-2056.
55 Chen J, Buchanan JB, Sparkman NL, Godbout JP, Freund GG, Johnson RW. Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain Behav Immun.2008; 22:301-311.
56 Godbout JP, Chen J, Abraham J, et al. Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. FASEB J.2005; 19: 1329-1331.
57 Kurosawa S, Kato M. Anesthetics, immune cells, and immune responses. J Anesth.2008; 22: 263-277.
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