【作者】 潘睿；

【导师】 常立文； Lee Wei-hua；

【作者基本信息】 华中科技大学 ， 儿科学， 2012， 博士

【摘要】 第一部分丙酮酸钠对缺氧缺血脑损伤的保护作用目的运用新生大鼠建立体外缺糖缺氧细胞和体内缺氧缺血脑损伤动物模型,观察丙酮酸钠(Sodium Pyruvate, SP)对新生大鼠缺氧缺血脑损伤的保护作用。方法模拟缺氧缺血建立细胞模型(oxygen and glucose deprivation, OGD)和动物模型(Hypoxia ischemia, HI)。结扎生后7天新生大鼠左侧颈总动脉,暴露于8%氧气2.5个小时,构建良好稳定的HI动物模型。提取新生大鼠大脑皮质原代神经元,体外培养7天,给与缺氧缺糖2.5个小时,构建OGD细胞模型。分别在不同时间点给予不同浓度的SP治疗。细胞试验分为对照组、OGD组和不同浓度SP治疗组,而动物模型分为假手术组、模型组和不同浓度、给药时间SP治疗组。LDH和MTT用于检测细胞死亡和生存状态。TTC染色、FJB染色、Cresyl Violet染色检测缺氧缺血脑组织损伤后不同时间点神经元的凋亡和脑损伤程度。结果体外实验显示,OGD后24小时SP可以逆转大脑皮层原代神经元LDH释放的增加和MTT水平的下降,且表现为剂量-依从性。2mM和4mM浓度效果最佳。HI后48小时脑组织TTC染色,SP在浓度125-1000 mg/kg之间可以显著降低HI对未成熟脑组织的损伤,如果在HI损伤后5分钟腹腔注射给与SP500 mg/kg可以获得最大疗效。和模型组相比,SP在HI损伤后24小时显著降低左侧(损伤侧)大脑皮层FJ-B染色阳性神经元。Cresyl Violet染色显示HI后第7天大脑冠状位形态发现丙酮酸钠可以使HI损伤后左侧存活／右侧存活的大脑皮层组织和海马组织比例分别从45.3士11.9%、39.2±8.7%升高到80士5.5%、67.3±9%。结论体外体内实验都显示丙酮酸钠对缺氧缺血脑损伤有神经保护作用第二部分丙酮酸钠对缺氧缺血脑损伤远期行为学的影响目的明确丙酮酸钠(Sodium Pyruvate, SP)能否促进新生大鼠缺氧缺血脑损伤后远期功能恢复。方法构建缺氧缺血(Hypoxic-ischemic, HI)动物模型,给予SP治疗。实验分为三组,假手术组、模型组和SP治疗组。HI损伤后3周进行足失误试验(Foot Fault Test, FFT),记录大鼠在5分钟内50步中右侧前肢及后肢失足次数,评价大鼠感觉运动功能。在HI造模后8周进行Morris水迷宫(Morris Water Maze, MWM)试验检测SP对大鼠空间学习记忆能力的影响。MWM试验包括隐蔽平台试验(Learning Trial) 4天和第5天的空间探索试验(Probe Trial)及可视平台试验(Cued Trial),记录大鼠的逃避潜伏期(Latency)、原平台象限停留时间(The Time Spent in the Platform Quadrant)及游泳速度(Velocity)。行为学试验后取大脑行Cresyl Violet染色观察大脑皮层(感觉运动功能)和海马(空间学习记忆能力)损伤情况。结果在FFT中,模型组(The Vehicle-treated Group)中大鼠右侧前、后肢失足次数明显高于假手术组(Sham Group),但SP组中失足次数明显下降,说明SP可促进脑损伤后大鼠感觉运动功能恢复。MWM隐蔽平台试验中,模型组大鼠逃避潜伏期明显高于假手术组,而SP治疗后大鼠逃避潜伏期明显被改善。空间探索试验中,模型组大鼠在原平台象限停留时间只有假手术组的一半,而SP治疗后大鼠停留时间明显升高,且三组大鼠游泳速度无明显差异,说明SP可改善脑损伤后大鼠空间学习记忆功能。可视平台试验结果三组之间无差异排除了视觉障碍对空间学习记忆影响。大鼠HI损伤后左侧存活／右侧存活的大脑皮层和海马组织分别为51%士9.9%和28.7%±7.4%,SP治疗后分别为87.2%±3.9%和77.9%士3.6%,两者相比有显著差异。结论SP可促进HI损伤后大鼠感觉运动功能和空间记忆功能的恢复第三部分丙酮酸钠在缺氧缺血脑损伤中神经保护机制的研究目的运用OGD细胞模型和HI动物模型,研究丙酮酸钠对缺氧缺血脑损伤神经保护作用的相关机制。方法分别与OGD和HI后不同时间点检测三磷酸腺苷(Adenosine Triphosphate, ATP)、活性氧自由基(Reactive Oxygen Species, ROS)、Bcl-2家族的促凋亡蛋白Bax、凋亡信号通路蛋白Cleaved Caspase-3和Caspase-3、存活信号通路蛋白P-Akt和Akt表达水平的变化,并选取OGD和HI后以上各指标有统计学差异的时间点给予SP治疗。细胞模型分为对照组、OGD组和SP治疗组,动物模型分为假手术组、模型组和SP治疗组。结果1.与对照组对比,OGD后ROS水平逐渐升高,呈时间依赖型,于4h开始升高有显著差异(p<0.05),24h升至最高(OGD/Ctrl=1.5±0.03, p<0.05)。SP将OGD后24h升高的ROS降低到了接近正常的水平(p<0.05)。2.和假手术组对比,HI后ROS水平于3h升至最高(p<0.05),后逐渐下降。SP将模型组HI后3h升高的ROS水平降低到了接近正常的水平(p<0.05)。3.与对照组对比,OGD后ATP水平于4h降至最低(OGD/Ctrl=0.49±0.07, p<0.05), SP将OGD后降低的ATP水平升高了约50%(p<0.05)。4.分别于HI后3h、16h给予SP治疗,SP分别将不同时间点模型组中降低的ATP水平升高约50%以上(p<0.05)。5.相比于对照组,OGD后0.5h, Bax表达水平达到最高(p<0.05),24h回到基础水平。SP将OGD后0.5h升高的Bax表达基本降到了对照组水平(p<0.05)。6.H1后3h,相比于假手术组,模型组中Bax显著升高(p<0.05),而SP治疗降低了HI后升高的Bax水平(p<0.05)。7.相比于对照组,OGD后Cleaved Caspase-3表达水平于4h逐渐升高(p<0.05),24h表达水平达到最高(p<0.05)。SP将OGD后24h升高的Bax表达水平降低约50%(p<0.05)。8.HI后24h,模型组中Cleaved Caspase-3显著升高(p<0.01),而SP治疗组中Cleaved Caspase-3水平与模型组相比降低了约50%(p<0.05)。9.相比于对照组,OGD后P-Akt表达水平先升高,于1h表达水平达到最高(p<0.01),后逐渐下降。OGD后1h,与OGD组对比,SP进一步升高了P-Akt表达水平(p<0.05)。10.HI后, P-Akt表达先升高后下降,于16小时降至假手术组的一半(p<0.05)。HI后16h,与模型组对比,SP治疗显著升高P-Akt的表达水平(p<0.05)。结论体内HI、体外OGD试验均显示,SP可能通过改善大脑能量代谢(ATP)、降低氧化应激水平(ROS)和阻止神经细胞死亡程序启动途径发挥缺氧缺血脑损伤的神经保护作用。更多还原

【Abstract】 PartⅠNeuroprotective Effect of Sodium Pyruvate on Hypoxic-ischemic Brain InjuryObjective:In this study, the protective effect of sodium pyruvate against hypoxia-ischemic brain injury was investigated in neonatal rats.Methods:Both in vivo (Hypoxia ischemia, HI) and in vitro (oxygen and glucose deprivation, OGD) models were established in this study.In vivo HI model employed unilateral (left/ipsilateral) carotid ligation in postnatal day 7 rats with exposure to 8% hypoxia for 2.5h. And in vitro OGD model employed primary cortical neurons on 7 DIV from neonatal rats subjected to oxygen and glucose deprivation for 2.5h. In vitro, cell death and cell viability were assessed using LDH and MTT kits respectively in OGD model. In vivo, Fluoro-Jade B (FJB) staining,2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining, cresyl violet (CV) staining were used to detect neuronal apoptosis and brain damage at different time points after HI injury.Results:In vitro study suggested LDH release increased and MTT levels decreased at 24 hours after OGD while SP reversed the effect in a dose dependent manner.2mM and 4mM rescued neurons from OGD induced injury to almost normal levels. In vivo study, SP at concentrations ranging from 125 to 1000 mg/kg markedly reduced hypoxic-ischemic injury to the immature brain and the maximal protection was achieved at 500 mg/kg if SP was administered at 5 min post-HI injury. Compared to the vehicle-treated group, FJB-positive neurons in the left/ipsilateral cortex at 24 h post-HI were significantly reduced by SP treatment. On brain morphology shown by cresyl violet staining, SP treatment increased the surviving brain tissues from 45.3±11.9% to 80±5.5% (cortex) and 39.2±8.7% to 67.3±9%(hippocampus).Conclusion:Both in vivo and in vitro studies showed SP took a neuroprotective effect on hypoxic-ischemic brain damage. PartⅡEffect of Sodium Pyruvate on Long-term Behavior After Hypoxic-ischemic Brain InjuryObjective:To determine whether SP can improve long-term functional recovery after hypoxic-ischemic brain injury on neonatal rats.Methods:The foot-fault test was performed at 3 weeks post-HI to assess sensorimotor function. Numbers of foot-faults with right forelimb and right hind limb per 50 steps was counted within 5 min. Morris Water maze (MWM) test were performed at 8 weeks post-HI to assess memory function. The learning trials are conducted over 4 days on rats. At the end of learning trials, a probe trial and a cued trial are given on day 5. Latency, the time spent in the platform quadrant and velocity were recorded. After MWM, tissue survival in the cortex and the hippocampus was quantified using cresyl violet staining.Results:In comparison to the sham group, rats in the vehicle-treated group showed significantly increased foot fault in right side at 3 weeks post HI. SP treatment resulted in a profound improvement in both right forelimb and hind limb foot faults. The latency in the vehicle-treated group was significantly increased, whereas SP treatment significantly reduced the latency. The time spent in the platform quadrant was reduced by 2 times in the vehicle-treated group (p<0.05) and SP treatment significantly increased the time spent in the platform quadrant (p<0.05). There is no significant difference in velocity among groups in the water maze test (p>0.05). The long-term effect of SP treatment significantly increased the cortical survival from 51%±9.9% in vehicle-treated to 87.2%±3.9% in SP-treated and the hippocampus survival from 28.7%±7.4% in vehicle-treated to 77.9%±3.6% in SP-treatedConclusion:SP improved sensorimotor and memory functions on rats subject to HI insult. PartⅢThe Mechanism of Neuroprotective Effects of Sodium Pyruvate on Hypoxic-ischemic Brain InjuryObjective:To study the relevant mechanism of neuroprotective effects of sodium pyruvate on Hypoxic-ischemic Brain InjuryMethods:ATP and ROS were measured to evaluate cerebral metabolism and oxidative stress both in vivo and in vitro. Bax, a pro-apoptotic signal in the outer mitochondrial membrane for immature neurons and cleaved Caspase-3, an apoptosis signal were detected both in vivo and in vitro. The expression of cleaved Caspase-3, total Caspase-3, phosphoralated Akt, total Akt, Bax were detected by western blot. A key survival signaling kinase, phosphoralated Akt was also tested.Results:Both in vivo and in vitro studies demonstrated that hypoxia-ischemic induced brain injury result in ROS accumulation and ATP depletion, and also increased Bax and cleaved caspase-3 expression, and decreased phosphoralated Akt. Sodium pyruvate attenuated hypoxia-ischemic induced brain injury by preventing an increase in intracellular reactive oxygen species (ROS) levels and maintaining ATP levels. Meanwhile, sodium pyruvate decreased Bax and cleaved caspase-3 expression, while activating P-Akt expression.Conclusion:Both in vivo and in vitro studies demonstrated SP protected neonatal brain from hypoxic-ischemic injury through maintaining cerebral metabolism and mitochondrial function.更多还原