【作者】 徐隋意；

【导师】 潘速跃；

【作者基本信息】 南方医科大学 ， 神经病学， 2013， 博士

【摘要】 神经保护剂的动物实验和人类临床试验结果差异较大。很多神经保护剂在各种动物缺血性模型中被证明有效,但是没有一个在人类Ⅲ期临床试验有作用。通过回顾性分析人类神经保护剂的随机对照试验(RCT)探讨神经保护剂临床转化失败的原因,我们发现很多因素与之相关：模型选择,麻醉剂选择,生理学指标监测,造模成功标准,栓子性质,再灌注损伤,梗死面积,治疗时间窗,药物渗透性,血药浓度,性别差异,结果评估等。而入院前“家庭药物”超早期治疗和多靶点治疗或许是未来考虑的方向。低温可以通过多靶点抑制缺血后损伤通路发挥神经保护作用。颈动脉内冰盐水灌注(ICSI)是最快速的低温诱导方式。我们在前期工作中发现,在大鼠缺血性卒中模型中低温的治疗时间窗比血管冲洗的作用时间宽。然而持续大量的ICSI来维持局部脑低温会导致病人一些列的副作用。因此我们考虑将这种特殊的治疗方法应用于未来临床时,我们意识到,为了在治疗效果和减少持续ICSI大容量盐水灌注的副作用方面权衡利弊,我们将其改良为间断ICSI。我们近期研究发现,与持续ICSI相比,间断策略可以维持更长时间的低温,对血细胞比容影响更小,可能为一种更有潜力的神经保护手段。虽然前期研究显示间断多次低温对大鼠大脑中动脉梗死(MCAO)模型有治疗效果。间断低温的神经保护机制仍不清楚。因此本实验利用胎鼠皮层神经元糖氧剥夺模型来体外模拟缺血性卒中,利用细胞模型高通量的特点比较不同低温模式对神经损害机制的影响,探讨间断多次低温可能的神经保护靶点。为筛选最优的间断低温模式以及未来临床应用提供理论依据。第一章皮层神经元原代培养胎鼠皮层神经元原代培养可以制作多种神经疾病的体外细胞模型。但是胎鼠脑体积较小,解剖难度相对较大,皮层神经元急性分离和培养操作环节复杂,至今尚未有统一的操作路径。因此探讨一种简便,合理,可重复性好的操作方法对于研究细胞模型至关重要。首先脱颈处死SPF级E18SD孕鼠,取出子宫和胎鼠。为了减少神经元代谢,保证其活力,接下来的解剖过程在碎冰上进行。我们将传统的断头后分离解剖顶端皮层改良为经鼻入路双侧对称分离法进行解剖,缩短了胎鼠皮层的解剖时间。然后用体视镜显微剥离皮层脑膜和血管,以减少脑膜细胞和血管细胞等混杂细胞对神经元纯度的干扰。由于这个过程时间较长,显微解剖在含有10%胎牛血清(FBS)和谷氨酰胺的HG-DMEM培养液的培养皿中进行以确保解剖过程中神经元的能量代谢。将剥离干净的胎鼠脑皮层置入含有冰HG-DMEM(不含FBS)的一次性3.5cm灭菌培养皿中,用灭菌的显微解剖剪剪碎至约1mm。考虑到传统的胰酶消化速度不好控制,我们用木瓜酶结合DNA酶I序贯消化。终止消化后,经过巴氏管吹打和细胞筛过滤得到神经元的单细胞悬液,用0.2%锥虫蓝染色以确保细胞团块要少于10%,死细胞数量小于1%。用0.1mg/ml左旋多聚赖氨酸预包被过的培养瓶以50000/cm2的密度接种神经元,4小时后发现大部分细胞已经贴壁。用neurobasal+B27+谷氨酰胺+青链双抗的培养液换液后继续培养。原代培养的神经元通过DAPI染核,B-3-TUBULIN骨架蛋白免疫荧光鉴定。神经元纯度通过阳性细胞计数和流式细胞仪测量均提示大于95%,可以满足进一步实验需要。第二章糖氧剥夺模型制作及低温干预方案缺血性卒中发生时,血液供应减少导致病灶区营养和氧气相应剥夺。这是形成“核心”梗死灶和缺血半暗带的重要原因。而这一过程可以用体外细胞卒中模型来进行模拟。核心梗死灶中已经死亡的神经元没有研究价值,而位于缺血半暗带的神经元是我们体外卒中模型研究的重点。越接近核心梗死灶,糖氧剥夺程度越重,而缺血半暗带区糖氧剥夺程度相对较轻。因此我们可以通过精确控制神经元培养液中的“糖”等营养成分来模拟糖剥夺。也可以通过厌氧培养箱来控制神经元培养环境中的“氧”浓度来模拟氧剥夺。相应的,如果缺血性卒中后期,责任血管发生再通或者是侧支循环建立恢复供血,又会面临着缺血再灌注损伤。而在体外细胞模型我们可以通过重新恢复神经元培养液和氧供给来研究这一重要的病理生理过程。本实验在神经元糖氧剥夺模型的基础上实施间断多次低温干预,具体方案为：糖剥夺用磷酸盐缓冲液(PBS)置换培养液,氧剥夺用厌氧培养手套箱进行(氧含量小于1%),糖氧剥夺时间为90分钟。低温干预包括持续低温组(CH)和间断多次低温组(IH)。低温组由33℃细胞培养箱(Thermo, USA)中进行,常温组设定为37℃(均为含5%CO2的细胞培养箱)。持续低温1组(CH1)的低温时长为6小时(与其他间断多次低温组的低温时间之和相同)。持续低温2组(CH2)的低温时长为12小时(与其他间断多次低温组的总时长相同)。间断低温1组(IH1)是1小时低温间隔1小时常温。间断低温2组(IH2)的低温间断周期为1.5小时。间断低温3组(IH3)的间断周期为2小时,观察指标的终点为低温干预后的48小时。同时设立正常对照组与糖氧剥夺(OGD)对照组。低温干预后我们接下来从各个角度评估其作用。第三章间断多次低温对糖氧剥夺神经元的保护作用本章旨在探讨间断多次低温对糖氧剥夺后神经元可能的保护靶点,以及比较持续低温和间断低温对其损伤抑制作用的不同特点。本章按照第一章方法原代培养胎鼠皮层神经元,然后按照第二章的方法制作糖氧剥夺模型以及实施低温干预。截至终点时观察各项指标。首先从宏观的角度比较各个组细胞形态学变化。然后通过神经元微环境代谢的角度,比较各组细胞活力的变化,检测细胞损伤释放入上清的酶标记物(神经元微管蛋白-2)以及兴奋性氨基酸(谷氨酸及其氧化酶)。接下来探测细胞内氧化损伤,酸中毒,钙超载,线粒体去极化等,探讨间断多次低温对糖氧剥夺后神经元是否具有保护作用以及其可能的机制。首先我们对各组间神经元形态学进行比较,正常组的神经元折光性强,胞体呈立体状,轴突生长旺盛,且与周围神经元相互联系呈网络状。而0GD组的神经元密度明显下降,神经元大量死亡漂浮导致数量明显减少。轴突崩解,华勒变性严重。而0GD后低温干预各组的神经元形态均恢复良好,仅有少量轴突断裂,与正常对照组相比,细胞形态学变化并不明显。由于单纯从神经元形态学判断低温的效果并不可靠,有些貌似“正常”的神经元微观已经发生了变化,接下来我们将从不同角度探索间断多次低温可能的作用。从神经元活力的角度上看,我们用Cell counting kit-8试剂盒(CCK-8)检测各组间神经元活力,结果显示CCK-8正常组(0.2984±0.0017),OGD组(0.2205±0.0215),CH1组(0.2617±0.0015),CH2组(0.2535±0.0052),IH1组(0.2329±0.0026),IH2组(0.2724±0.0033),IH3组(0.2814±0.0025)。统计分析后提示与正常对照组相比,糖氧剥夺后神经元活力下降。在持续低温组中,6小时持续低温与12小时持续低温均有助于恢复糖氧剥夺后神经元活力,其中6小时持续低温效果好于12小时持续低温。在间断低温组中,1小时间断低温虽然有活力恢复的趋势,但并未达到统计学意义。1.5小时和2小时间断低温均有助于恢复糖氧剥夺后神经元活力,其中2小时间断低温效果优于1.5小时间断低温。间断低温与持续低温相比,1.5小时和2小时间断低温效果均优于持续低温组,其中2小时间断低温效果最佳。从神经元酶损伤标记物的角度上看,我们用大鼠神经元微管相关蛋白-2(MAP-2)酶联免疫吸附(ELISA)试剂盒来检测上清中的MAP-2。结果显示正常组(1.0780±0.1366),OGD组(1.3461±0.0966),CH1组(1.1858±0.0881),CH2组(1.2893±0.0747),IH1组(1.3251±0.0616),IH2组(1.3325±0.1618),IH3组(1.1808±0.1593)。统计分析后提示糖氧剥夺后神经元微环境中的MAP-2含量增加。在持续低温组中,6小时持续低温可以抑制糖氧剥夺后MAP-2的释放,12小时持续低温有下降的趋势但无统计学差异。在间断低温组中,2小时间断低温可以抑制糖氧剥夺后MAP-2的释放,1小时和1.5小时间断低温有下降的趋势,但未达到统计学意义。间断低温与持续低温相比,2小时间断低温和6小时持续低温对减少OGD后MAP-2释放的效果相当。从兴奋性氨基酸谷氨酸及其氧化酶的角度上看,我们用Amplex(?) Red谷氨酸/谷氨酸氧化酶试剂盒检测各组间神经元上清中谷氨酸和谷氨酸氧化酶活性。结果显示正常组(2698.91±206.74),OGD组(2719.35±195.53),CH1组(2763.77±227.90),CH2组(2703.73±278.95),IH1组(2697.83±203.14),IH2组(2714.32±195.63),IH3组(2653.49±±230.16)。谷氨酸氧化酶正常组(478.62±52.08),OGD组(490.14±70.19),CH1组(494.33±92.01),CH2组(450.42±54.26),IH1组(462.52±±95.59),IH2组(468.53±33.50),IH3组(455.95±±46.10)。统计显示,各组间数据无统计学差异。通过分析原因,我们发现是由于终点的设置错过了检测时间窗所致,提示我们微透析早期动态监测谷氨酸释放或许更为合理。由此可见从神经元微环境代谢的角度或许不能灵敏的反应神经元内部的变化。接下来我们用荧光探针的技术探讨间断多次低温对细胞内部靶点的影响。在此之前,我们首先要明确间断多次低温是否对糖氧剥夺神经元具有保护作用。我们用Hoechst33342对OGD后神经元进行定性染色,发现正常神经元核染成淡蓝色,而另外有部分细胞核被染成了范围小,蓝色荧光强度高的亮点,提示确实发生了凋亡。于是我们用TUNEL法定量检测各组间神经元凋亡,结果显示正常组为(6676.2±91.84),OGD组(8327.6±177.19),CH1组(7524.8±310.71),CH2组(6092.4±85.09),IH1组(4121±124.83),IH2组(7570±118.53),IH3组(5628.20±699.82)。经统计分析提示,与正常对照组相比,糖氧剥夺后48小时,神经元凋亡明显增加。在持续低温组中,6小时持续低温可以抑制糖氧剥夺后凋亡发生,但效果不及12小时持续低温。所有间断低温组均可以有效抑制神经元凋亡。其中,1小时间断低温的保护效果优于1.5小时间断低温,但与2小时间断低温无统计学差异。间断低温与持续低温组相比：1小时间断低温效果优于6小时和12小时持续低温,1.5小时间断低温的保护效果与6小时持续低温相当,但不如12小时持续低温。2小时间断低温效果优于6小时持续低温,与12小时持续低温的保护效果相当。综合以上结果分析提示1小时间断低温对糖氧剥夺后神经元凋亡保护效果最佳。由此可见,间断多次低温确实可以减少糖氧剥夺后神经元凋亡。前期我们通过回顾性分析发现,氧化损伤,酸中毒,钙超载,线粒体衰竭是神经元受损的主要通路。接下来我们将通过荧光探针对其相应的细胞内靶点进行检测。活性氧的产生是氧化损伤的主要原因之一,我们用DCFH-DA荧光探针测量细胞内活性氧(ROS)发现,正常组(397.67±49.34),OGD组(1954±69.94),CH1组(424.67±21.36),CH2组(395.33±33.47),IH1组(562.67±92.79),IH2组(331±26.06),IH3组(8098.33±1033.02)。统计分析提示,糖氧剥夺后48小时,神经元内活性氧显著增加。在持续低温组中,6小时持续低温和12小时持续低温均能抑制糖氧剥夺后神经元内活性氧产生,两者的效果无差别。在间断低温组中,1小时,1.5小时间断低温均能抑制糖氧剥夺后活性氧产生,且二者的保护效果没有差别。2小时间断低温数据变异较大,谨慎起见,暂不纳入分析。间断低温与持续低温抑制糖氧剥夺后活性氧的效果相当。超氧阴离子自由基的产生也是氧化损伤的重要因素,我们用Dihydroethidium(DHE)荧光探针测量神经元内超氧阴离子自由基发现,正常组(69.4±3.36),OGD组(167.75±15.59),CH1组(64.2±2.28),CH2(114.4±7.54),IH1(43.6±2.30),IH2(52.8±1.79),IH3(52.6±1.14)。经统计分析提示,糖氧剥夺后神经元内超氧阴离子自由基含量增加。在持续低温组中,6小时和12小时持续低温均可以抑制超氧阴离子自由基产生。在间断低温组中,1小时,1.5小时和2小时间断低温也同样可以制糖氧剥夺后神经元内超氧阴离子自由基产生,其中1小时间断低温效果最好,1.5小时和2小时间断低温的保护效果没有差别。间断低温与持续低温相比,间断低温组减少OGD后超氧阴离子自由基的效果均优于持续低温组,其中1小时间断低温效果最佳。我们用BCECF AM荧光探针,测量细胞内pH变化。其中正常组(994.2±58.87),OGD组(67.8±24.45),CH1组(92.2±10.26),CH2组(104.2±8.14),IH1组(70.8±43.3),IH2组(90.0±51.87),IH3组(84.4±12.86)。统计分析提示,糖氧剥夺后神经元内pH水平明显下降。持续低温与间断低温虽有恢复OGD后神经元pH的趋势,但均无统计学意义的改善。我们用FLUO-3AM荧光探针测量细胞内游离钙,由于同一批次胎鼠皮层神经元培养数量有限,为保证样本的同质性,我们首先测量糖氧剥夺组和低温各组的平均荧光强度。由于不同批次的神经元荧光变异非常大,不具有可比性。所以接下来利用另外一批次的神经元补充测量正常组和糖氧剥夺组。具体数据如下,OGD组(1706.8±40.3),CH1组(2586.8±97.91),CH2组(741.4±13.46),IH1组(797.4±12.82),IH2组(403.8±15.61),IH3组(688.2±13.22)。补充测量正常组(58±1.58),OGD组(84.2±1.64)。统计分析提示,糖氧剥夺后神经元游离钙含量升高。在持续低温组中：6小时持续低温不能降低神经元糖氧剥夺后细胞内游离钙水平,12小时持续低温有效。在间断低温组中：1小时,1.5小时和2小时间断低温均可以降低神经元糖氧剥夺后细胞内游离钙水平,其中1.5小时效果最佳,2小时间断低温的保护效果优于1小时间断低温。间断低温与持续低温组相比,1小时间断低温抑制OGD后细胞内钙超载的效果不如12小时持续低温,1.5小时间断低温和2小时间断低温的效果优于12小时持续低温,其中1.5小时间断低温抑制糖氧剥夺后神经元内钙超载的效果最佳。我们用JC-1荧光探针测量线粒体膜电位变化发现,JC-1正常组(0.21±0.03),OGD组(1.85±0.16),CH1组(2.23±0.23),CH2组(0.98±0.05),IH1组(1.15±0.14),IH2组(0.61±0.14),IH3组(0.93±0.05)。统计分析提示,糖氧剥夺能损害线粒体膜电位去极化。在持续低温组中,6小时持续低温不足矣抑制线粒体膜电位改变,12小时持续低温有效。在间断低温组中,1小时,1.5小时和2小时间断低温均可以保护糖氧剥夺后线粒体膜电位。且它们之间的保护作用相同。间断低温与持续低温组相比,1小时,1.5小时和2小时间断低温对糖氧剥夺后神经元线粒体膜电位改变的保护效果同12小时持续低温效果相当。总之,本实验发现间断多次低温可以有效抑制胎鼠皮层神经元0GD后凋亡。间断多次低温发挥神经保护作用的机制为抑制OGD后活性氧及超氧阴离子自由基产生,减轻细胞内钙超载,保护线粒体膜电位损伤。虽然持续低温与间断多次低温均具有神经保护作用,但间断多次低温是一种更具临床可行性的神经保护策略。更多还原

【Abstract】 The discrepancy in results for neuroprotective agents in animal experiments compared to clinical trials is a major problem. While many neuroprotective agents have been proven effective in a variety of animal ischemic stroke models, none of them have been shown to work in phase III clinical trials. We retrospectively summarizes the neuroprotectants selected for human randomized controlled trials (RCT) and explores the reasons behind the clinical translational failure of these agents. Here, we suggest that there are many factors (model selection, anesthetic choice, physiological monitoring, model success criteria, embolus property, reperfusion damage, infarction area, therapeutic time window, drug penetration, blood concentration, gender difference, outcome evaluation) responsible for this phenomenon. Ultra-early treatment using a "home run" drug and multi-target therapy may be the most promising for future consideration.Hypothermia is the most effective way of neuroprotection through inhibiting multi-target mechanism of ischemic injury. Intracarotid cold saline infusion (ICSI) is the fastest hypothermia induction. Therapeutic time window of hypothermia is broader than cerebral artery flushing in carotid saline infusion after transient focal ischemic stroke in rats. Continuous ICSI to maintain local brain hypothermia is unrealistic in clinic for massive infusion fluid volume. Intermittent ICSI can greatly reduce the difficulties in liquid capacity management and brain temperature fluctuation. Compared with traditional ICSI, the intermittent method has a longer duration of hypothermia and less effect on hematocrit and offers more potentially improved neuroprotection.Although primary studies showed that intermittent hypothermia did work on MCAO models in rats. However, the neuroprotective mechanisms of the intermittent multiple hypothermia following ischemic stroke was still unknown. The fetal rat cortical neurons after an oxygen glucose deprivation (OGD) in vitro was established to mimic ischemic stroke cellular model. The high throughput of cell model is used to investigate the the possible neuroprotective mechanisms of intermittent hypothermia for screening optimal intermittent hypothermia pattern, as well as a theoretical basis for future clinical applications.Chapter one. Primary culture of cortical neuronsThe primary culture of fetal rat cortical neurons is widely used in cell models of many neurological disorders. However, owing to the complicated procedures involved in dissection and culture, a universally accepted protocol for their derivation has not yet been determined. Diverse techniques make it difficult to readily compare results obtained from different cell models and to repeat experiments in other laboratories. Therefore, it is essential to develop a simple and reproducible protocol for the study cell models. SPF-class E18Sprague Dawley pregnant rats were sacrificed by cervical dislocation. The uterus was removed by rotating clockwise from the left lower quadrant. The placenta was removed, and the color, fetal movements and number of stillbirths were recorded. In order to reduce the metabolism of neurons and guarantee its vitality, the following anatomical process was carried out on crushed ice. We improved traditional microscopic anatomy path to bilateral symmetry separation via nasal approach on E18SD fetal rats. The next was to dissect the skull and dura mater. Take care when removing the pia mater and blood vessels to reduce the interference of the meningeal and vascular cells. Owing to the long process, microscopic anatomy was in a petri dish with cold HG-DMEM containing10%fetal bovine serum (FBS) and glutamine to ensure the energy metabolism. Clean-stripped fetal rat cortex was put into a3.5cm petri dish containing cold FBS-free HG-DMEM and then cut into1mm with sterilized scissors. We have modified the procedure to a sequential digestion of papain and DNase I for taking into account the fast traditional trypsin digestion. Cell suspension was harvested by pipetting and cell sieve, and stained by trypan blue to ensure the dead was less than1%and the clum was less than10%. The neurons were cultured on0.1mg/ml LPP-precoated vessels with the density of50000per cm2. The most neurons were adhered after4hours when the culture medium was replaced by neurobasal with B27and glutaminate. Nearly all the cells were DAPI-and β-tubulin Ⅲ-positive. The determination of neuronal purity using dark field imaging suggests that the percentage of β-tubulin Ⅲ-immunostained neurons was over95%; accordingly96.8%were identified as neurons when assessed by flow cytometry.Chapter two. OGD model and hypothermia interventionThe decrease in blood supply caused OGD in focal zone after an ischemic stroke. It is the important reason to form "core" infarction and ischemic penumbra. And this process can be simulated in vitro via cell model. The dead neurons in core infarction can not be saved, while the ones in ischemic penumbra still have chance. We focus the latter one. The closer to the core of the infarct, the heavier oxygen-glucose deprivation, but relatively mild in ischemic penumbra zone. So we can precisely control the sugar and other nutrients in culture medium to simulate glucose deprivation. Anaerobic incubator was used to control the oxygen concentration to mimic oxygen deprivation. when responsible vascular recanalization or collateral circulation restores the blood supply, it will face ischemia-reperfusion injury. We can restore neuronal culture medium and oxygen supply to study this important pathophysiological process. Therefore, intermittent hypothermia was administrated on neuronal OGD model. Specific programs are as follows:Glucose deprivation in the culture solution was replaced with phosphate buffered saline (PBS). Oxygen deprivation was applied by an anaerobic culture glove box, oxygen-glucose deprivation time was90minutes. There are7groups in the experiment:normal group, OGD group, continuous hypothermia1group (CHI), continuous hypothermia2group (CH2), intermittent hypothermia1group (IH1), intermittent hypothermia2group (IH2) and intermittent hypothermia3group (IH3). Hypothermia intervention contained continuous hypothermia (CH) and intermittent hypothermia (IH). Hypothermia was provided by33℃cellular incubator (Thermo, USA) while the normothermia by37℃(Both cellular incubators were supplied with5%CO2). The hypothermia runtime of CHI group was6hours which were consistent with the sum of hypothermia runtime in other IH groups. The hypothermia runtime of CH2group was12hours which were consistent with the total time in other IH groups. IH1group ran1-hour hypothermia and1-hour normothermia alternately. The intermittent cycle of IH2group was1.5hour, and IH3group was2hours. The endpoint was48hours later after hypothermia intervention, while the normal and OGD groups were served as controls. The effects of hypothermia were assessed from different angles afterwards. Chapter three. The neuroprotective mechanisms of the intermittent hypothermia on OGD neuronsThis chapter aims to explore the potential targets of intermittent multiple hypothermia and compare the differences between CH and IH. Fetal rat cortical neurons were cultured as chapter one, OGD and hypothermia phase as chapter two. The indicators were observed at the endpoint. Cell morphology between each groups were observed under a inverted microscope (Olympus, Japan) with bright field. By the neuronal microenvironmental metabolism angle, we compared the cell viability change, enzyme-labeled substance of cell injury, and excitatory amino acids released into the supernatant in each group. Intracellular acidosis, calcium overload, oxidative damage, mitochondrial depolarization, and apoptosis were detected then.First, we compared the morphology of neurons in each group. Normal neurons refracted three-dimensional shape, cell body, axon growth was strong, and with the surrounding interconnected network shape. In OGD groups, neuronal density decreased significantly for dead floating and appeared axon disintegration or Waller degeneration. The neurons recovered well in hypothermia groups, only a small number of axons fractured. Compared with the normal control group, the morphological changes were not obvious. The microstructure of some seemingly "normal" neurons has changed. Next we will seek for the potential evidence of intermittent hypothermia.Judging from the point of view of the neuronal vitality detected by CCK-8kit, the data was as follows:normal group (0.2984±0.0017), OGD group (0.2205±0.0215), CH1group (0.2617±0.0015), CH2group (0.2535±0.0052), IH1group (0.2329±0.0026), IH2group (0.2724±0.0033), IH3group (0.2814±0.0025). Neuronal vitality drops after OGD. In continuous hypothermia, both6-hours and12-hours continuous hypothermia were helpful to restore the vitality of neurons after OGD.6hours pattern was better than12hours. In intermittent hypothermia,1-hour intermittent pattern did not work.1.5-hour and2-hours intermittent hypothermia were helpful to restore the vitality of neurons after OGD.2-hours intermittent hypothermia was better than1.5-hour intermittent hypothermia. Comparison of intermittent and continuous hypothermia:except for1-hour intermittent hypothermia, the1.5-hour and2-hours intermittent hypothermia were more effective than continuous hypothermia,2-hours intermittent hypothermia was the best.From the point of view of neuronal enzyme injury markers, we used a rat neurons microtubule associated protein-2(MAP-2) enzyme-linked immunosorbent assay (ELISA) kits to detect the supernatant MAP-2. The data was as follows:normal group(1.0780±0.1366), OGD group (1.3461±0.0966), CHI group (1.1858±0.0881), CH2group (1.2893±0.0747), IH1group (1.3251±0.0616), IH2group (1.3325±0.1618), IH3group (1.1808±0.1593). Statistical analysis indicated that neuronal microenvironmental MAP-2level increased after OGD. In continuous hypothermia,6-hours continuous hypothermia could inhibit MAP-2release after OGD,12-hours continuous hypothermia had no such effect. In intermittent hypothermia,2-hours intermittent hypothermia could inhibit MAP-2release after OGD,1-hour and1.5-hour intermittent hypothermia had no such effect. Comparison of intermittent and continuous hypothermia, the neuroprotection of2-hours intermittent hypothermia and6-hours continuous hypothermia was same.From the point of view of the excitatory amino acid glutamate and its oxidase, we detected the glutamic acid and glutamate oxidase activity in the supernatants with the Amplex(?) Red kit. The results showed glutamic acid in normal group (2698.91±206.74), OGD group (2719.35±195.53), CHI group (2763.77±227.90), CH2group(2703.73±278.95),IH1group(2697.83±203.14), IH2group(2714.32±195.63), IH3group (2653.49±230.16). Glutamate oxidase in normal group (478.62±52.08), OGD group (490.14±70.19), CH1group (494.33±92.01), CH2group (450.42±54.26), IH1group (462.52±95.59), IH2group (468.53±33.50),IH3group (455.95±46.10). There was no difference between groups. By analyzing the reason, we found that it due to the settings on the endpoint which missed detection time window. The early dynamic monitoring of glutamate release with microdialysis may be more reasonable. Thus, the microenvironmental metabolism changes may not be sensitive to response inner neuronal changes. Next, we will use the technology of fluorescent probes to explore the interior of the cell targets after the intermittent hypothermia.Prior to this, we must first clear whether intermittent hypothermia was protective on oxygen-glucose deprivated neurons. Qualitative stained with Hoechst33342after OGD neurons found that normal nucleus was calamine blue but the apoptosis showed small high-lighted blue. Quantitative detection of each group was finished by TUNEL kit, the data was as follows:normal group (6676.2±91.84), OGD group (8327.6±177.19), CHI group (7524.8±310.71), CH2group (6092.4±85.09), IH1group (4121±124.83), IH2group (7570±118.53), IH3group (5628.20±699.82). Compared with the control group, neuronal apoptosis increased significantly after48hours of90minutes oxygen-glucose deprivation. In continuous hypothermia,6-hours continuous hypothermia could reduce neuronal apoptosis after OGD but the effect was weaker than12-hours. In intermittent hypothermia, all the intermittent hypothermia reduced neuronal apoptosis after OGD.1-hour and2-hour intermittent hypothermia were better than1.5-hour pattern. Comparison of intermittent and continuous hypothermia,1-hour intermittent pattern was more effective than6-hour and12-hour continuous hypothermia. The neuroprotection of1.5-hour intermittent pattern was same as6hour, but weaker than12hour.2-hour intermittent hypothermia was better than6hour, but same as12hour. Based on the above,1-hour intermittent hypothermia was the most effective way to reduce apoptosis after OGD. Our retrospective analysis found that, oxidative damage, acidosis, calcium overload, mitochondrial failure were the main pathways of the damage to the neurons. Next, we will test the corresponding intracellular targets.The generation of reactive oxygen species is the main reason of oxidative damage. DCFH-DA fluorescent probe measurement of intracellular reactive oxygen species (ROS) found normal group (397.67±49.34), OGD group (1954±69.94), CHI group(424.67±21.36),CH2group(395.33±33.47),IH1group(562.67±92.79), IH2group(331±26.06), IH3group(8098.33±1033.02). Statistical analysis indicated that, neuronal reactive oxygen species (ROS) had a significant increase at48hours after OGD. In continuous hypothermia, both6-hours and12-hours continuous hypothermia could inhibit ROS after OGD, and the effectiveness was same. In intermittent hypothermia, both1-hour and1.5-hour intermittent hypothermia could inhibit ROS after OGD, The effectiveness of former two were same. The data of2-hours intermittent hypothermia varied greatly, it was not included in analysis. Based on the above, the effectiveness of intermittent hypothermia and continuous pattern were same.The generation of superoxide anion radical oxidative damage was also an important factor, we used dihydroethidium (DHE) fluorescent probe to detect superoxide anion radical. The data was as foloows:normal group (69.4±3.36), OGD group (167.75±15.59), CHI group (64.2±2.28), CH2group (114.4±7.54), IH1group (43.6±2.30), IH2group (52.8±1.79), IH3group (52.6±1.14) Intraneuronal superoxide anion radical increased after OGD. In continuous hypothermia, both6-hours and12-hours continuous hypothermia did work. In intermittent hypothermia, all the intermittent hypothermia could inhibit superoxide anion radical after OGD,1-hour pattern was best, and the rest1.5-hour or2-hours pattern were same on effectiveness. Comparison of intermittent and continuous hypothermia, all the intermittent hypothermia groups were better than continuous hypothermia,1-hour intermittent hypothermia performed best.We used BCECF AM fluorescent probe to measure intracellular pH changes. The data was as follows:normal group (994.2±58.87), OGD group (67.8±24.45), CHI group (92.2±10.26), CH2group (104.2±8.14), IH1group (70.8±43.3), IH2group (90.0±51.87), IH3group (84.4±12.86). Intra-celluar pH level decreased significantly. The pH level appeared recovery trend after both continuous and intermittent hypothermia, but there was no statistically significant improvement.FLUO-3AM fluorescent probe was applied to measure intracellular free calcium. Due to the limited amount of the same batch of fetal rat cortical neurons, we firstly measured the mean fluorescence intensity of the oxygen-glucose deprivated group and the hypothermia groups to ensure the sample homogenicity. The neuronal fluorescence of different batches varied largely, the results were not comparable. So another batch of neuronal supplemental measure was applied to test normal group and OGD group. The data of first batch were as follows:OGD group (1706.8±40.3), CHI group (2586.8±97.91), CH2group (741.4±13.46), IH1group (797.4±12.82), IH2group (403.8±15.61), IH3group (688.2±13.22). The supplemental-measure results were as follows:normal group (58±1.58), OGD group (84.2±1.64). Intraneuronal free calcium level increased after OGD. In continuous hypothermia,6-hours continuous hypothermia had no effect on decreasing cellular free calcium after OGD, while12-hours pattern worked. In intermittent hypothermia, all the intermittent hypothermia could decrease cellular free calcium after OGD. The1.5-hour intermittent hypothermia is the best. Comparison of intermittent and continuous hypothermia,1-hour intermittent hypothermia was less effective than12-hours continuous hypothermia.1.5and 2-hours pattern was prior to12-hours continuous hypothermia.1.5-hour intermittent pattern was the most effective on inhibiting calcium overload after OGD.JC-1fluorescent probe was applied to measure mitochondrial membrane potential changes. The data was as follows:normal group(0.21±0.03), OGD group (1.85±0.16), CHI group (2.23±0.23), CH2group (0.98±0.05), IH1group (1.15±0.14), IH2group (0.61±0.14), IH3group (0.93±0.05). Oxygen-glucose deprivation can lead to the mitochondrial membrane potential depolarization. In continuous hypothermia,6-hours continuous hypothermia was not enough to inhibit mitochondrial membrane potential depolarization, but12-hours pattern worked. In intermittent hypothermia, all the intermittent hypothermia could be helpful, and the effectiveness was same. The intermittent hypothermia was as effective as12-hours continuous pattern.In conclusion, the study found that intermittent hypothermia could inhibit apoptosis of fetal rat cortical neurons after OGD. The neuroprotective mechanisms were as follows:inhibiting ROS and superoxide anion radical generation, alleviating intracellular calcium overload, protecting mitochondrial membrane potential damage. Both continuous hypothermia and intermittent hypothermia had a neuroprotective effect, but intermittent hypothermia was a potentially clinical strategy.更多还原