【作者】 刘江勤；

【导师】 陈超；

【作者基本信息】 复旦大学 ， 新生儿， 2009， 博士

【摘要】 第一部分凋亡在三日龄大鼠缺氧缺血脑损伤恢复期的作用以及远期预后目的:围产期损伤后神经细胞持续凋亡是新生儿缺氧缺血性脑损伤（hypoxicischemic brain injury,HIBI）远期预后不良的机制之一,目前对于损伤后恢复期神经细胞凋亡的作用和机制研究较少。本研究通过3日龄大鼠缺氧缺血脑损伤模型,评估大脑损伤的细胞类型和凋亡基因在损伤后的持续表达,并通过免疫组织学、影像学和行为学评估大鼠成年期预后。方法:通过结扎右侧颈总动脉合并缺氧（6-7%）2.5小时,建立3日龄SD大鼠缺氧缺血脑损伤模型（HI组n=52,对照组n=41）。应用Oligo GEArray^?凋亡基因芯片比较脑损伤后12小时与损伤后7天凋亡基因表达的差异;应用免疫组织化学染色检测肿瘤坏死因子相关的凋亡诱导配体（TRAIL）及其死亡受体、Caspase-3在脑组织中的表达;大鼠成年后在42日龄进行大脑磁共振成像检查,44日龄采用Morris水迷宫测试其学习和空间记忆能力,48日龄进行MBP染色评估大脑髓鞘化。结果:①3日龄大鼠缺氧缺血脑损伤后,未成熟的神经元和少突胶质细胞均发生损伤。损伤后恢复期（HI后7天）细胞凋亡基因进一步激活,包括TNF及其受体家族、Bcl 2家族、Caspase家族,以及募集Caspase结构域和死亡诱导效应因子。②损伤后24小时和72小时脑组织TRAIL的死亡受体DR5主要表达在大脑皮层的板下层锥形神经元,缺氧缺血脑损伤显著增加DR5阳性细胞数（n=5,24小时:HI组右侧、左侧和对照组分别为130±62、72±17和35±20,F=6.54,p=0.013,两两比较HI右侧与左侧比较,p＜0.05;72小时分别为147±67、85±30和58±15,F=4.71,p=0.033,两两比较HI右侧与对照组比较p＜0.05）;损伤后72小时,HI组右侧大脑皮质和脑室周Caspase-3阳性细胞数显著多于对照组右侧（286±61和171±63,n=4,t=2.613,p＜0.05）。③42日龄时头部磁共振显示HI组大鼠右侧大脑皮质ROI值变异度与对照组没有显著性差异。HI组右侧皮质体积较左侧和对照组显著减小（分别为84±15.1、130.9±17.3和133.0±10.9[mm³],F=25.3,p＜0.001）。④水迷宫实验HI组上台花费时间明显较对照组长（第4日:52.71±35.87与17.80±8.85[s],p＜0.01）,上台成功率也明显低于对照组（第4日:60%与98.33%,p＜0.001）。HI组大鼠在训练第二日在各项指标上均有提高,但其后的训练不能改善其表现。在第5日记忆测试中,虽然HI大鼠游泳的距离与对照组没有差异,其经过站台的次数显著少于对照组（0.3±0.5比1.1±0.8次,t=3.28,p＜0.05）。缺氧后12小时表达NG2和04的未成熟少突胶质细胞数明显减少,而第48日龄时脑组织MBP染色显示HI组右侧大脑髓鞘化明显低于对侧和对照组同侧。结论:①3日龄大鼠缺氧缺血脑损伤后,损伤的细胞包括未成熟的神经元和少突胶质细胞;②损伤后恢复期（HI后7天）细胞凋亡基因进一步激活,包括TNF及其受体家族、Bcl-2家族、Caspase家族,以及募集Caspase结构域和死亡诱导效应因子;③缺氧缺血损伤调节TRAIL受体DR5、Caspase-3在神经元上的表达,可能参与调节损伤后细胞的归宿;④早期发生缺氧缺血脑损伤引起脑组织坏死和激活凋亡基因,导致皮质萎缩,影响动物的空间学习和记忆功能。第二部分N-乙酰半胱氨酸对窒息新生猪血流动力学和神经细胞凋亡的影响目的:目前新生儿缺氧缺血脑损伤尚缺乏令人满意的治疗方法,特别是在损伤后的恢复期,针对损伤的多种机制的策略可能具有较好的临床应用前景。N-乙酰半胱氨酸（NAC）具有清除氧自由基、抗炎和抗细胞凋亡的作用,但在缺氧缺血脑损伤恢复期的神经保护作用尚缺乏相应的研究。本项目通过足月新生猪窒息模型,观察N-乙酰半胱氨酸治疗窒息新生猪大脑氧供和血供以及神经细胞凋亡的影响。方法:通过机械通气给予11-14%氧气建立新生猪（n=8）脑缺氧-再氧合损伤模型,新生猪在缺氧2小时后给予N-乙酰半胱氨酸（NAC,150 mg/kg推注,然后20 mg/kg/h静脉注射维持24小时）治疗24小时,同时给予100%氧气1小时,观察缺氧损伤后48小时内心率、血压、颈总动脉血流（CCAF）和氧供（CCADO₂）,以及脑组织内一氧化氮（NO）浓度的变化;并采用ELISA法检测缺氧损伤后48小时右侧皮质脂质过氧化物（LPO）和凋亡标记物Caspase-3的活性。设缺氧-再氧合对照组（n=8）和假手术组（n=6）。结果:新生猪在发生缺氧后,动脉血pH显著下降并伴有严重的代谢性酸中毒（pH平均为7.05,HCO₃平均为10-11 mM）,给予100%氧气后,缺氧NAC治疗组和对照组的pH和HCO₃均迅速恢复正常。缺氧-再氧合结束后早期,NAC治疗迅速增加颈总动脉血流和氧供（NAC组、对照组和假手术组,CCAF分别13.53±1.20、9.70±1.12和17.17±1.31 ml/min/kg,F=66.93,p＜0.001;CCDO₂分别为0.53±0.23、0.48±0.16和1.85±0.36 mL O₂/mL/kg,F=62.70,p＜0.001）。但其后48小时观察期间NAC对颈总动脉血流和氧供没有进一步的改善。NAC治疗在早期不影响心率和平均动脉压,但在缺氧后24小时NAC治疗组新生猪的心率显著高于对照组（NAC组、对照组和假手术组分别为228±41、191±28和188±23次/分,F=3.738,p＜0.001）。在缺氧后NAC治疗组和对照组脑组织皮质内NO浓度显著高于假手术组,并持续整个48小时观察期,NAC治疗不影响脑组织内NO浓度。48小时后与缺氧-再氧合对照组比较,NAC治疗显著减少新生猪右侧大脑皮质中LPO（NAC组、对照组和假手术组分别为229±41、639±152和280±40 uM,F=5.13,p＜0.05）和Caspase-3活性（NAC组、对照组和假手术组分别为0.4±0.1、0.8±0.1和0.6±0.1 U/mg,F=4.28,p＜0.05）。结论:新生猪缺氧-再氧合损伤后48小时内,给予NAC静脉注射治疗能改善大脑血流动力学,迅速恢复大脑的血流和氧供。这种作用不依赖组织内的一氧化氮调节,而可能与其减少脑组织的氧化应激和神经细胞的凋亡有关。第三部分进展性气胸对新生猪大脑血流动力学和血气的影响目的:气胸是NICU常见的并发症,严重的气胸影响机体的血流动力学和减少大脑的血供和氧供,是神经系统预后不良的高危因素。但在气胸进展过程中对大脑血流供应的影响缺少研究。通过建立足月新生猪气胸模型,观察气胸在进展过程中对机体血气、生理指标以及颈总动脉氧供和血供的影响。方法:新生猪（n=9）给予机械通气后,在20分钟内每5分钟给予右侧胸腔注射10ml/kg（共40 ml/kg）空气建立进展性气胸的模型。观察心率、平均动脉压、中心静脉压、颈总动脉血流（CCAF）和颈总动脉氧供（CCADO₂）,以及动脉血气的变化。另有7只新生猪作为对照组。结果:随着气胸的进展,胸腔内注射20ml/kg空气后SaO₂和PaO₂显著下降（20ml/kg时间点与基础值,SaO₂分别为83.5±12.4和94.4±4.0;PaO₂分别为52.5±12.4和77.5±14.9,p＜0.05）,而血气pH和PaCO₂并没有发生改变。在注射30ml/kg空气后中心静脉压和颈总动脉血流显著上升（与基础值比较,CVP分别为7.5±3.7和6.1±2.9mmHg;CCAF分别为43±18和35±14 mL/min/Kg,p＜0.05）,但CCADO₂显著下降（与基础值比较分别为2.5±1.8和3.6±1.3mL O₂/min/Kg,p＜0.05）,而气胸进展对心率和血压没有显著的影响。结论:气胸进展的早期,在发生代谢性酸中毒前,尽管颈总动脉血流升高,颈总动脉的氧供由于低氧血症而显著下降。中等程度的气胸不影响新生猪的心率和血压,但其中心静脉压逐渐增高。更多还原

【Abstract】 PARTⅠ:Further activation of apoptosis during the recovery of the hypoxic ischemic brain injury of 3-day old rat pups and the outcome in adulthoodObjectives:Although the persistent apoptosis is one of the important factors of poor longterm outcome of newborn infants with hypoxic ischemic（HI） brain injury after perinatal events,little information is available regarding the mechanism of the activation of apoptosis. We aimed to compare the differences of the activation of apoptotic genes between the acute and recovery stages after HI brain injury in a 3-day-old-rat HI brain injury model,to observe the expression of apoptotic proteins on the neural cells,and to investigate myelination of the brain,changes in cranial MRI and cognitive function in young adulthood.Methods:A 3-day-old SD rat HI brain injury model was established by right carotid artery ligation followed by 2.5 hours of hypoxia（6%O₂）（HI group,n=52）.Control pups were sham-operated（n=41）.Right brain cortex was collected at 12 hours（acute stage） and 7 days （recovery stage） after HI and subjected to an apoptosis Oligo GEArray^?,and the differences between 12 hours and 7 days after HI were analyzed.The expression of TRAIL and its receptors,Caspase-3 in the cerebral cortex and periventricular white matter regions was examined at 24 and 72 hours after HI using immunochemistry staining.MRI scanning, Morris water maze test and myelination assessment were performed in both groups at 42,44 and 48 days of age,respectively.Results:Comparing to 12 hours after HI,the apoptotic genes were further activated at 7 days after HI in the brain of HI rat pups,including TNF,Caspase,Bcl 2,Caspase Recruitment Domains,Death Domain and Death Effector Domain families.Although there were no changes in the expression of TRAIL and its other receptors,HI was disclosed to enhance the expression of DR5 in the pyramidal neurons in the subplate of cortex at 24 and 72 hours after HI（24 hours after HI:130±62 vs.72±17 and 35±20 cells/mm² in right,left side of HI group and right cortex of control group,respectively;n=5,F=6.54,p＜0.05,1-way ANOVA,SNK, right vs.left of HI,p＜0.05;72 hours after HI:147±67 vs.85±30 and 58±15 cells/mm², F=4.71,p＜0.05,SNK,right of HI vs right of Control,p＜0.05）.Meanwhile,the expression of Caspase-3 in the cortex and periventricular white matter was found to be elevated at 72 hours after HI compared to those in control group（286±61 vs.171±63 cells/mm²,n=4, t=2.613,p＜0.05）,with no change at 24 hours.Furthermore,the MRI assessment demonstrated that the volume of right cortex of HI rats（n=8） was significantly smaller than that of left side and controls（n=6）（84±15.1 vs.130.9±17.3 and 133.0±10.9[mm³],F=25.3, p＜0.001,SNK,right cortex of HI vs.left and control,p＜0.05）.During the four days of navigation trial in the Morris water maze test,the HI rats demonstrated longer escape latency than the rats of control group（fourth day:52.71±35.87 vs 17.80±8.85[s],p＜0.01）.They crossed the platform less than those in the control group as well（0.3±0.5 vs 1.1±0.8 times, t=3.28,p＜0.05） in Space probe trial.The myelination with myelin basic protein staining was impaired in the right white matter of HIBI rats compared to the left corresponding region and the right side of control group at 48 days of age.Conelusions:The activation of apoptotic genes induced by HI persist upto 7 days,involving intrinsic and extrinsic apoptotic pathways.The apoptosis of neural cells leads to poor development of the cortex and myelination,and might compromise the memory and learning function in the young adulthood in rats with neonatal HI.PARTⅡ:N-Acetylcysteine improves hemodynamics and reduces oxidative stress in the brain of newborn piglets with hypoxia-reoxygenation injuryObjectives:Satisfied therapy of hypoxic ischemic brain injury（HIBI） of newborns needs to be developed with promising strategy on the different mechanisms of the damage.N-acetylcysteine （NAC）,a free radical scavenger with anti-inflammatory and anti-apoptotic features,was proven to minimize HIBI in various acute models,but the effects of NAC on the brain during recovery from HIBI needs to be further explored.Using a sub-acute swine model of neonatal hypoxia-reoxygenation（H-R）,we evaluated the long-term beneficial effect of NAC against oxidative stress and apoptosis.Methods:Newborn piglets were mechanical ventilated by a pressure-controlled ventilator via tracheostomy.The animals were randomly assigned into a sham group（without H-R,n=6） and two H-R experimental groups（n=8 each） with 2 hours normocapnic alveolar hypoxia and 1 hour 100%O₂ of reoxygenation followed by 0.21 of FiO₂ for 47 hours.Five minutes after reoxygenation,hypoxic piglets received either normal saline（H-R controls） or NAC （150 mg/kg bolus and 20 mg/kg/h i.v.for 24 hours） in a blinded randomized fashion. Hemodynamic changes and blood gas variables were recorded throughout H-R.The activity of cortical caspase-3 and lipid hydroperoxide（LPO） of the fight hemisphere of the piglets was detected after 48 hours of observation.Results:After hypoxia,the arterial pH decreased significantly with metabolic acidosis developed（mean arterial pH～7.05;mean arterial HCO₃ 10-11 mM）.Upon reoxygenation, both arterial pH and HCO₃ recovered similarly in both H-R groups.Treating the piglets with NAC significantly increased both carotid blood flow（CCAF） and oxygen delivery during the early phase.Even though there was no difference between these two H-R groups thereafter, both CCAF and carotid oxygen delivery of the H-R group remained lower than the sham groups throughout the experimental period.Compared with H-R controls,significantly higher amount of anesthetics and sedative was required to maintain the NAC-treated piglets in stable condition throughout the experimental period,indicating a stronger recovery.Post-resuscitation NAC treatment also significantly attenuated the increase in cortical caspase-3 and LPO levels.Conclusions:In newborn piglets with H-R insults,post-resuscitation administration of NAC reduces cerebral oxidative stress with improved cerebral oxygen delivery,probably through anti-apoptosis mechanism. PartⅢ:Sequential Changes of Hemodynamics and Blood Gases in Newborn Piglets with Developing PneumothoraxObjectives:Pneumothorax,a common complications of newborns hospitalized in NICU,is a high risk of major neurological disabilities due to the attenuation of cerebral perfusion and oxygen delivery in severe situation.But little information is available regarding the temporal changes in hemodynamics and blood gases during the development of a moderate pneumothorax in a neonate.Thus,we investigated the temporal changes of hemodynamics and arterial blood gases in a neonatal swine model of unilateral pneumothorax in this prospective observational controlled animal research.Methods:Experimental pneumothorax（n=9） was created by intermittent progressive introduction of 10 ml/Kg of room-air every 5 min to a total 40 ml/Kg,via a 20G Insyte^? angiocatheter placed in the fourth intercostal space in line with the fight frontal limb. Changes in heart rate,mean arterial pressure,central venous pressure（CVP）,common carotid arterial flow（CCAF） and arterial blood gases were measured and compared with the normoxic baseline and a control group（n=7）（ANOVA）.Results:As the pneumothorax developed,CVP increased alter injecting 30 ml/Kg of roomair （p＜0.001 vs.baseline and control） with no significant changes in heart rate and mean arterial pressure.After 20 ml/Kg of air was introduced,arterial blood gases showed deteriorating oxygenation.CCAF increased and carotid oxygen delivery declined after 30 ml/Kg（p＜0.05 vs.baseline and control）.Conclusions:Deterioration in oxygenation was noted early in the development of pneumothorax in newborn piglets followed by metabolic acidosis.CVP progressively increased despite the lack of significant changes in systemic hemodynamics when moderate pneumothorax developed.Although CCAF increased during a moderate pneumothorax, carotid oxygen delivery decreased.更多还原