树鼩脑局部血栓性缺血的扩布性抑制机制探讨

目的:观察树鼩血栓性脑缺血时,对侧神经元血小板活化因子(PAF)受体活化和神经元线粒体呼吸功能改变,探讨皮层扩布性抑制的可能机制。方法:采用光化学法诱导脑血栓形成,分别观察脑缺血对侧神经元超微结构、脑细胞膜PAF受体、单胺氧化酶(MAO)活性、单胺类递质含量以及神经元线粒体呼吸功能。结果:树鼩脑缺血时对侧皮层淤血,神经元线粒体肿胀;脑缺血4h对侧神经元PAF受体亲和量降低,其变化以24 h最为显著(P<0.01);缺血区线粒体Ⅲ态呼吸速率、呼吸控制率(RCR)及氧化磷酸化效率(P/O)均明显降低;缺血对侧皮层的P/O降低(P<0.05),Ⅲ态呼吸速率明显抑制(P<0.01);伴随着对侧脑组织MAO活性的升高(P<0.01),对侧皮层的去甲肾上腺素(NA)和5-羟色胺(5-HT)含量降低而5-羟吲哚乙酸(5-HIAA)含量明显升高(P<0.01)。结论:树鼩脑缺血时对侧皮层神经元PAF受体的活化以及MAO活性的增强可能在扩布性抑制的发生中具有重要作用。     

树鼩血栓性脑缺血中心区及半暗区神经元PAF受体结合特性的研究

目的：观察血栓性局部脑缺血过程中缺血中心区及半暗区血小板活化因子（PAF）受体的消长变化,探讨PAF在脑缺血中心区及半暗区神经元继发性脑损伤中的分子机制。方法：建立光化学诱导树鼩血栓性局部脑缺血模型并提取树鼩脑细胞膜蛋白,用[³H]-PAF放射配体结合试验检测中枢神经细胞膜不同特性的PAF结合位点（受体）。结果：树鼩脑细胞膜上存在两种亲和性不同的PAF受体,即高亲和性和低亲和性受体,其亲和力（kD）分别为(3.61±0.72) nmol/L（kD₁）和17.04±2.41) nmol/L（kD₂）相应的最大结合容量（B_max）分别为(1 457.94±168.01) pmol/g蛋白和(5 017.40±742.16) pmol/g蛋白。脑缺血4、24及72 h中心区、半暗区及对侧区高、低亲和性受体的kD值、B_max值均显著低于假手术组（P<0.01）,中心区及半暗区尤为明显,其中以缺血后24 h的变化最为显著。结论：PAF受体在介导缺血性脑损伤过程中起着重要作用,缺血中心区及半暗区机能代谢的不同与PAF受体亲和特性及最大结合容量改变不同有关,亦是PAF介导继发性脑损伤的重要分子基础。     

缺血后适应减轻树鼩缺血性脑水肿及脑梗死的机制

目的 观察缺血后适应对树鼩血栓性脑缺血时大脑皮层脑水含量、局部脑血流、梗塞面积及神经元超微结构的影响,探讨其对树鼩脑缺血时神经保护的可能机制。 方法 将88只健康成年树鼩随机分为对照组、脑缺血4 h组、脑缺血24 h组、后适应4 h组及后适应24 h组（每组n=8）,另取8只动物做HE染色（n=3）及电子显微镜观察（n=5）。本实验采用光化学反应诱导树鼩血栓性脑缺血而建立脑缺血动物模型,在脑缺血模型建成后4 h夹闭缺血侧颈总动脉5 min,再灌注5 min,如此交替进行3个循环以建立缺血后适应模型。测定大脑皮层局部脑血流,脑组织含水量,脑梗死范围,并观察皮层及海马CA1区神经元超微结构改变。 结果 脑缺血时神经元固缩,线粒体肿胀,嵴溶解或形成空泡,内质网肿胀,内质网池形成。缺血后适应能使海马CA1区神经元固缩减少,线粒体和内质网的病理改变减轻,细胞水肿改善。随着缺血时间的延长,缺血24h组脑水含量明显增加86.81%±1.08%,此时脑梗塞面积明显扩大33.00%±3.03%,局部脑血流明显降低（134.27±28.75）ml/min。缺血后适应24h组脑组织含水量明显减少（81.04%±1.04%,P＜0.01）;脑梗塞面积缩小（16.79%±1.29%,P＜0.01）;而局部脑血流明显增加［（195.25±21.18）ml/min,P＜0.01］。 结论 缺血后适应可缓解树鼩缺血性脑水肿并缩小梗死范围,其机制可能与改善局部脑血流有关。     

树鼩脑缺血半暗区PAF受体介导的微环境改变及GB保护机制的探讨

目的研究树鼩血栓性脑缺血时,血小板活化因子(PAF)受体活化介导的缺血半暗区微环境改变,并探讨PAF受体拮抗剂—银杏内酯B(ginkgolide,GB)的神经保护机制。方法采用光化学诱导树鼩血栓性局部性脑缺血模型,用3H?PAF放射免疫标记法检测缺血区微环境脑细胞PAF受体亲合性(Kd值)和结合特性(Bm ax值)的变化;用密度梯度法及原子吸收分光法分别测定缺血微环境水含量及Na+、Ca2+含量,并于光化学反应后6 h静注GB(5 mg/Kg),观察其对脑血栓形成后24 h时缺血微环境的改善效应。结果树鼩脑血栓形成后缺血半暗区微环境的改变以24 h为著,脑细胞膜高亲和性、低亲和性PAF受体的Kd值及Bm ax值明显降低,其中Kd1及Bm ax1分别为(0.611±0.9)nM和(419.4±72.6)fmol.mg-1蛋白,Kd2及Bm ax2分别为(4.08±0.5)nM和(676.8±98.66)fmol.mg-1蛋白(与对照组相比P<0.01);GB可促使缺血微环境脑细胞PAF受体Kd及Bm ax的恢复,并具有改善局部脑水肿和缓解Ca2+超载(P均<0.01)。结论PAF受体活化在介导缺血半暗区微环境改变中具有重要作用,GB可改善缺血半暗区微环境和逆转PAF受体活化介导的神经元泵功能障碍。     

CsA对树鼩海马微灌流谷氨酸和钙所致线粒体应激的影响

目的：观察环孢菌素A(CsA)对树鼩海马由谷氨酸（Glu）及钙（Ca²⁺）引起微环境改变所致线粒体应激的影响，并探讨其分子机制。方法:单泵等速微灌流系统行树鼩海马Glu及Ca²⁺微灌流，24 h后免疫组化法检测海马神经元细胞色素C（Cyt C）蛋白表达；低温差速离心分离海马神经元线粒体和胞质部分，免疫印迹（Western blotting）法检测Cyt C在胞内表达空间分布；实时荧光定量PCR技术检测海马caspase-3及caspase-9 mRNA的含量。微灌流Glu和Ca²⁺溶液后6 h于舌下iv CsA 40 mg/kg BW，24 h后观察上述指标的改变。结果:树鼩海马微灌流Glu和Ca²⁺溶液后24 h，海马神经元Cyt C表达增强，而线粒体Cyt C含量显著下降，同时胞质部分可见Cyt C；海马组织caspase-3、caspase-9mRNA明显升高；微灌流后6 h静脉注射CsA组, 海马神经元Cyt C表达显著减少，而线粒体Cyt C含量则显著增加，胞质部分未见Cyt C;海马组织caspase-3、caspase-9 mRNA降低。结论:海马微环境中Glu与Ca²⁺的大量堆积,可促进线粒体Cyt C释放，激活caspase级联反应而导致线粒体应激；CsA的神经保护效应可与其抑制线粒体通透性转导孔（MPT）开放，防止Cyt C释放及减少caspase-3和caspase-9的活化有关。     

正常和缺血大鼠脑线粒体对环孢菌素A的反应

目的：研究正常和缺血脑线粒体对环孢菌素A（CsA）的反应，并观察线粒体ATP敏感性钾通道与线粒体渗透性转换孔的关系。方法：本实验采用分光光度法，在分离线粒体上观察线粒体渗透性转换孔抑制剂和线粒体ATP敏感性钾通道开放剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体，0.5 μmol/L和 1 μmol/L CsA以及30 μmol/L 二氮嗪（DE）均可明显减轻Ca²⁺诱导的线粒体肿胀程度，苍术苷（Atr）能取消此作用，而5 μmol/L CsA不能减轻线粒体的肿胀。在全脑缺血5 min后的线粒体，0.5 μmol/L CsA可减轻Ca²⁺诱导的线粒体肿胀，该作用可被Atr取消，但1 μmol/L CsA不能减轻肿胀；30 μmol/L DE也可明显减轻Ca2+诱导的缺血脑线粒体肿胀程度，100 μmol/L和200 μmol/L 5-羟基癸酸盐（5-HD）和Atr均可取消其作用。结论：缺血脑线粒体对线粒体渗透性转换孔开放的抑制剂比正常脑线粒体更为敏感，脑线粒体ATP敏感性钾通道激活可能是抑制线粒体渗透性转换孔开放的调控机制之一。     

JAK2-STAT3信号通路在树鼩脑缺血后适应中的作用机制

 目的：观察JAK2-STAT3信号转导通路在树鼩缺血后适应（ischemic postconditioning,IPoC）神经保护中的调控作用,探讨阻断JAK2-STAT3通路后脑损伤加重的机制。方法：通过光化学反应建立树鼩血栓性脑缺血模型;于缺血后4 h夹闭患侧颈总动脉3次（每次5 min）实施IPoC。于IPoC前10 min侧脑室注射AG490（JAK2抑制剂）后,采用TTC染色观察树鼩脑梗死面积的变化,通过HE染色和电镜观察脑皮层神经元形态改变及超微结构变化,应用Western blot检测IPoC及AG490处理后皮层t-STAT3和p-STAT3蛋白水平的变化。结果：缺血24 h,皮层神经元固缩,线粒体肿胀,嵴溶解;脑梗死面积占半脑面积的（24.78±3.30）%;此时皮层神经元STAT3磷酸化水平明显增高（P<0.01）。IPoC后皮层神经元损伤减轻,线粒体肿胀改善,脑梗死面积占半脑面积的百分比减小为（17.67±1.83）%（P<0.01）,STAT3磷酸化水平进一步增高（P<0.01）。然而,给予AG490处理后,皮层神经元损伤加重,脑梗死面积再次增大为（23.85±2.77）%（P<0.05）,STAT3磷酸化水平则明显降低（P<0.05）。结论：IPoC可能通过调控STAT3的磷酸化而减轻树鼩缺血性脑损伤,抑制JAK2-STAT3信号通路可抵消IPoC的保护效应而加重脑损伤。     

高血糖和脑缺血对树鼩皮层不同区域VEGF表达的影响

目的：研究高血糖及局灶性脑缺血条件下,树鼩皮层不同区域VEGF表达的变化,探讨脑缺血、高血糖与VEGF之间的相互关系。方法：用链脲佐菌素复制树鼩高血糖模型,并建立光化学诱导皮层局灶性脑缺血,观察缺血4 h、24 h及72 h的病理形态学改变并计数海马神经元密度,用免疫组化法测定上述时间树鼩缺血中心区、半暗带、对侧皮层VEGF表达的动态变化。结果：形态学观察显示,光化学反应后4 h照射区皮层可见梗塞灶;24 h病损达高峰;72 h伴随胶质细胞增生等修复性反应。相应时点高血糖加缺血组的损伤大于缺血组,以缺血后24 h(P<0.01)和72 h(P<0.05)尤为显著。免疫组化染色表明,缺血后4 h皮层缺血半暗区可见VEGF表达增加, 24 h达高峰,72 h减弱;单纯高血糖也使VEGF表达上调;高血糖加缺血组VEGF表达强于单纯高血糖组(P<0.05),但高血糖加缺血组与缺血组的同期值比较,无显著差异。结论：(1)在低等灵长类动物树鼩体内注射链脲佐菌素,并结合血栓性局部脑缺血方法学的应用能成功复制出实验性高血糖及脑缺血模型;(2)实验证明高血糖对局灶性脑缺血有恶化加重作用;(3)脑缺血及高血糖均可分别作为独立因素诱导VEGF的表达;但缺血与高血糖相加对VEGF表达未显示出叠加效应。     

缺血后适应调控树鼩脑缺血海马CA1区Akt（pS473）和Akt（pT308）过磷酸化的抗凋亡机制

观察树鼩脑缺血海马神经元Akt（pS473）和Akt（pT308）磷酸化改变对CA1区细胞凋亡的影响,探讨缺血后适应（PC）抑制细胞凋亡的可能机制。方法 用光化学诱导法诱导树鼩脑缺血并建立缺血PC模型;用免疫组织化学TACS原位凋亡检测试剂盒检测皮层及海马CA1区神经元凋亡数量,用免疫组织化学法检测Akt（pS473）和Akt（pT308）表达的空间分布,用ELISA法检测海马CA1区神经元Akt（pS473）和Akt（pT308）磷酸化水平;用电子显微镜观察其神经元超微结构改变。 结果 脑缺血后神经元皱缩,核消失以缺血24h为著。脑缺血后4 h、24 h及72 h皮层及海马CA1区神经元TUNEL阳性细胞数明显增加（P<0.01）,海马CA1区神经元Akt (pS473) 和Akt (pT308)的表达及磷酸化水平明显升高,以脑缺血4 h的改变最明显,分别为(152.3±3.5) units/mg、(130.8±2.6) units/mg和(149.5±4.7)units/mg和 (42.35±2.49) units/mg、(19.23±1.41) units/mg和(23.38±1.32) units/mg (P<0.01）。缺血PC组神经元损伤明显减轻,皮层及海马CA1区TUNEL阳性细胞明显减少（P<0.01）,且与海马神经元Akt (pT308)活化水平呈平行改变(P<0.05）。结论 树鼩脑缺血海马CA1区细胞凋亡与Akt(pS473) 和Akt (pT308)过磷酸化的信号机制有关,缺血PC抑制海马神经元Akt(pS473) 和Akt(pT308)双磷酸化可能具有抗凋亡作用。     

缺血后适应对树鼩脑缺血信号转导及转录激活因子3过磷酸化的调控作用

目的观察信号转导及转录激活因子3(STAT3)磷酸化在树鼩脑缺血后适应(PC)神经保护中的作用,并探讨其可能机制。方法将50只健康成年树鼩随机分为对照组、脑缺血4 h组、脑缺血24 h组、后适应4 h组和后适应24 h组(每组n=5),其中10只动物做HE染色(n=5)及电子显微镜观察(n=5)。本实验通过光化学反应建立树鼩血栓性脑缺血模型;于缺血后4 h夹闭患侧颈总动脉3次(每次5 min)实施缺血PC。采用TTC染色观察树鼩脑梗死面积的变化,通过HE和电子显微镜观察脑皮质和海马组织学改变及其超微结构变化,应用Western blotting检测皮层总STAT3(t-STAT3)及磷酸化STAT3(p-STAT3)蛋白表达变化。结果脑缺血后皮层血管内皮细胞肿胀,皮层及海马神经元损伤,线粒体肿胀、嵴溶解,以缺血24 h损伤最为明显,脑梗死面积达到(24.78±2.06)%。而皮层p-STAT3蛋白表达随缺血时间延长呈增高趋势,缺血4 h p-STAT3蛋白表达明显增高(0.24±0.1,P<0.01),缺血24 h p-STAT3蛋白表达则持续增高(0.32±0.1,P<0.01)。缺血PC处理后皮层血管内皮细胞水肿好转,皮层及海马神经元损伤减轻,脑梗死面积减小为(17.67±1.90)%(P<0.01)。与缺血组相比,缺血PC 4 h p-STAT3蛋白表达进一步升高(0.41±0.09,P<0.01),缺血PC 24 h p-STAT3蛋白表达增高更加显著(0.70±0.11,P<0.01)。结论树鼩脑缺血可导致STAT3磷酸化代偿性增强,缺血PC的脑保护作用可能与其促进STAT3过磷酸化有关。     

The effects of monobromobimane on neuronal cell death in the hippocampus after transient global cerebral ischemia in rats

Calcium accumulation and free radical formation in the mitochondria are suggested to result in opening of the mitochondrial permeability transition pore that may be an initial step in neuronal cell death. The purpose of the present study was to determine whether monobromobimane (MBM) was a possible protective agent against neuronal cell death after transient global ischemia and the swelling of isolated hippocampal mitochondria. Infusion of MBM (1 or 3 microg) to cerebral ventricles 30 min before ischemia attenuated the expression of TUNEL-labeled cells and neuronal cell death in the hippocampal CA1 region at 72 h of reperfusion dose-dependently. Treatment with MBM inhibited an increase in caspase-3-like activity at 48 h of reperfusion in the hippocampus. MBM (30-300 microM) also inhibited an enhanced swelling rate induced by Ca2+ and phenylarsineoxide in the isolated hippocampal mitochondria. These results suggest that in vivo treatment with MBM may protect against neuronal cell death through inhibition of the mitochondrial swelling and caspase-3-dependent apoptotic pathway.     

Possible mechanisms of Cyclosporin A ameliorated the ischemic microenvironment and inhibited mitochondria stress in tree shrews’ hippocampus

Objective: The ischemic brain damage is always accompanied by the significant accumulation of glutamate and calcium ions (Ca²⁺). Our objectives were to observe the effects of glutamate and Ca²⁺ overloading in tree shrew's hippocampal microenvironment on mitochondrial stress resulting in cytochrome C release and caspase apoptotic gene activation, and to explore the possible mechanism of Cyclosporin A (CsA) inhibiting mitochondrial stress. Methods: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. The extracellular contents of amino acidic neurotransmitters and Ca²⁺ were determined, respectively, with high performance liquid chromatography (HPLC) and atomic absorption spectrophotometry at 4, 24 and 72 h after cerebral ischemia. The glutamate–calcium chloride solutions were microperfused into hippocampus by a kind of single-pumped push–pull perfusion (SPPP) system under three-dimensional orientation instrument in tree shrews. At 24 h, the expression of cytochrome C was observed in perfused lateral hippocampus by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blot. Real time fluorescence polymerase chain reaction was used to evaluate the relative amounts of caspase-3 and caspase-9 mRNA. In the treated group, CsA (40 mg/kg) was intravenously injected at 6 h after the microperfuse or cerebral ischemia. The glutamate–calcium solutions were perfused into the hippocampus and inspected the above-mentioned items at 24 h. Data were compared between the two groups (ischemia group vs. sham group, or ischemia group vs. CsA group). Results: Thrombotic cerebral ischemia led to significant increase in extracellular glutamate and Ca²⁺ level of hippocampus (P < 0.01). The cerebral ischemia group and the microperfusion group, which cytochrome C immunoreactivity increased and Western blot analysis demonstrated that the cytochrome C content in the mitochondria of hippocampal cells decreased (P < 0.01), but the cytochrome C in the cytosol increased (P < 0.01). When CsA was intravenously injected at 6 h after the microperfusion or cerebral ischemia, the cytochrome C expression weakened and its release was diminished to a lesser extent. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate–calcium chloride solution perfused group. CsA treatment cut down the contents of caspase-3 mRNA and caspase-9 mRNA (P < 0.01). Conclusions: It is a primary factor that glutamate and Ca²⁺ accumulate in hippocampal microenvironment, which results in proapoptotic protein cytochrome C release from mitochondria into cytoplasm and caspase cascade activation, and finally mitochondria stress and neuronal secondary injury appear. The neuroprotection of CsA is in relation to inhibiting glutamate receptor overactivation and reducing the Ca²⁺ influx, which can decrease cytochrome C release and caspase mRNA transition.     

The neuroprotection conferred by activating the mitochondrial ATP-sensitive K+ channel is mediated by inhibiting the mitochondrial permeability transition pore

In order to further explore the mechanisms by which activation of mitochondrial ATP-sensitive potassium channels (mitoKATP) confers neuroprotection, we investigated the role of the mitochondrial permeability transition pore (MPTP) in in vivo and in vitro models. Adult male Sprague-Dawley rats were exposed to 90 min of middle cerebral artery occlusion (MCAO) followed by reperfusion for 22 h, when neurological scores and infarct volumes were evaluated. Activating mitoKATP by infusion of 2 mmol/L diazoxide into the ventricles 20 min before MCAO or inhibiting the MPTP by infusion of 1 micromol/L cyclosporin A 15 min before reperfusion significantly increased functional score and reduced infarction volume. Subsequent intracerebroventricular infusion of 2 mmol/L atractyloside, the MPTP opener, 10 min before reperfusion significantly attenuated the neuroprotective effects of diazoxide and cyclosporin A. The swelling of mitochondria isolated from brain was evaluated by spectrophotometry and served as a measure of MPTP opening. In isolated mitochondria, 100 micromol/L atractyloside attenuated the decrease of mitochondrial swelling induced by 30 micromol/L diazoxide or cyclosporin A (0.5 or 1 micromol/L). Furthermore, 100 micromol/L diazoxide or 1 micromol/L cyclosporin A both attenuated the fluorescence intensity in isolated mitochondria loaded with rhod-2 acetoxymethylester, and 100 micromol/L atractyloside abolished the effects of diazoxide and cyclosporin A. These results suggest that activation of mitoKATP protects the brain against injury, and this is probably mediated by attenuating mitochondrial Ca2+ overload and thus inhibiting MPTP opening during brain ischemia and reperfusion.     

Mannosylated liposomal cytidine 5' diphosphocholine prevent age related global moderate cerebral ischemia reperfusion induced mitochondrial cytochrome c release in aged rat brain

Mitochondrial dysfunctions generating from cerebral ischemia-reperfusion exert a potential threat on neuronal cell survival and hence, accelerate the aging process and age dependent neuropathology. Thirty min moderate cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) followed by 30 min reperfusion caused an increased diene production, depleted glutathione (GSH) content, reduced superoxide dismutase (SOD) and catalase activities and pyramidal neuronal loss in young (2 months old) and aged (20 months old) rat brain compared to sham operated controls. Cytidine 5' diphosphocholine (CDP-Choline) is a known neuroprotective drug. CDP-Choline after metabolism in the liver suffers hydrolysis and splits into cytidine and choline before entering systemic circulation and hardly circumvents blood brain barrier (BBB) as such. Previous reports show CDP-Choline liposomes significantly increased in vivo uptake compared to "free drug" administration in cerebral ischemia. To enhance the therapeutic concentration build up in brain we sought to formulate mannosylated liposomal CDP-Choline (MLCDP) utilizing the mannose receptors. We tested the therapeutic supremacy of MLCDP over liposomal CDP-Choline (LCDP) in global moderate cerebral ischemia reperfusion induced neuronal damage. CDP-Choline in MLCDP delivery system was found potent to exert substantial protection against global moderate cerebral ischemia reperfusion induced mitochondrial damage in aged rat brain. Membrane lipid peroxidation, GSSG/GSH ratio and reactive oxygen species (ROS) generation in cerebral tissue were found to be higher in aged, compared to young rat. Further decline of those parameters was observed in aged rat brain by the induction of global moderate cerebral ischemia and reperfusion. MLCDP treatment when compared to free or LCDP treatment prevented global moderate cerebral ischemia-reperfusion induced mitochondrial damage as evident ultra structurally and release of cytochrome c (cyt c) from mitochondria into cytosol and protected mitochondria to restore its normal structure and functions.     

Prevention of in vitro and in vivo acute ischemic neuronal damage by (2S)-1-(4-amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl) phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate (SUN N8075), a novel neuroprotective agent with antioxidant properties

(2S)-1-(4-Amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl) phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate (SUN N8075) is a novel antioxidant with neuroprotective properties. We examined whether SUN N8075 inhibited the neuronal damage resulting from permanent focal cerebral ischemia, and examined its neuroprotective properties in vivo and in vitro mechanism. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice, and the resulting infarction, brain swelling, and neurological deficits were evaluated after 24 h or 72 h. Brain damage was assessed histochemically using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and antibody recognizing 4-hydroxynonenal histidine adduct (4-HNE). In the in vitro study, we examined the effects of SUN N8075 on 1) lipid peroxidation in mouse brain homogenates and 2) cell viability and caspase-3 protease activity under a hypoxic insult or FeSO(4) in rat cultured cerebrocortical neurons. SUN N8075 administered either 10 min before or at 1 h after the occlusion reduced both infarction size and neurological deficits. SUN N8075 reduced brain swelling when administered 10 min before, 1 h, or 3 h after occlusion. Furthermore, only pretreatment (administered 10 min before) decreased infarct volume and brain swelling at 72 h after middle cerebral artery occlusion. SUN N8075 reduced the number of TUNEL-positive cells and decreased the level of oxidative damage, as assessed by immunopositive staining to 4-HNE. SUN N8075 inhibited lipid peroxidation, leakage of lactate dehydrogenase, caspase-3 activation induced by in vitro hypoxia, and the neuronal damage induced by in vitro FeSO(4) exposure. These findings indicate that SUN N8075 has neuroprotective effects against acute ischemic neuronal damage in mice and may prove promising as a therapeutic drug for stroke.     

银杏内酯B对树鼩血栓性脑缺血后GFAP表达的影响及机制探讨

目的:观察树鼠句血栓性脑缺血形成后不同部位星形胶质细胞表达胶质纤维酸性蛋白(GFAP)的时间消长改变,以及血小板活化因子(PAF)受体拮抗剂银杏内酯B(GB)对GFAP表达的影响,并探讨其可能机制。方法:建立光化学诱导树鼠句血栓性脑缺血模型,用免疫组化法检测缺血后4、2 4、72hGFAP表达,最后用图像分析系统测定其平均灰度。结果:脑缺血后半暗区GFAP表达增多,以2 4h最为显著,其平均灰度值为6 0 .33±3 .0 9(P <0 . 0 1) ,72h表达仍高,其平均灰度值为6 0. 88±2 . 6 2 (P <0 . 0 1) ,此时对侧及远隔区GFAP表达增强。光化学反应后6h于舌下静脉注射GB(5mg/kg) ,发现缺血后2. 4h半暗区星形胶质细胞GFAP表达明显下调,与对照组相比有显著差异(P <0 . 0 5 )。结论:缺血性脑损伤后星形胶质细胞表达GFAP增多与神经元受损有关;GB通过拮抗血小板活化因子(PAF)对神经元的损伤作用使星形胶质细胞表达GFAP减少。     

Increased mitochondrial DNA oxidative damage after transient middle cerebral artery occlusion in mice

Oxidative stress and DNA oxidation play important roles in the induction of ischemic neuronal cell death. However, the subcellular source of oxidized DNA detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) after ischemia has not been clarified although it is known to increase in the brain after ischemia. One-hour transient ischemia of the middle cerebral artery was induced in mice utilizing an intraluminal filament. The occurrence of superoxide anion as an ethidium (Et) signal, 8-OHdG, cytochrome c release and neuronal cell death were examined using immunohistological and biochemical techniques in sham-operated control (0h) and 1, 3, 6, 24, or 96h after reperfusion. Et signals were prominent in the cortical neurons of ipsilateral hemisphere 3h after reperfusion. Strong 8-OHdG immunoreactivity was observed 3-6h after reperfusion. Immunoassays after cell fractionation revealed a significant increase of 8-OHdG in mitochondria 6h after reperfusion. Immunohistochemistry revealed that the 8-OHdG immunoreactivity colocalized with a neuronal marker, microfilament 200 and a mitochondrial marker, cytochrome oxidase subunit I. Cytochrome c rose in cytoplasm at 6h and TUNEL-positive neurons noted 6-24h after ischemia. The present results suggest the possibility that the mitochondrial damage including mitochondrial DNA oxidation might be responsible for the induction of ischemic neuronal cell death.     

Resveratrol pretreatment protects rat brain from cerebral ischemic damage via a sirtuin 1–uncoupling protein 2 pathway

Resveratrol is a natural polyphenol found in grapes and wine and has been associated with protective effects against cardiovascular diseases. In vitro, both resveratrol preconditioning (RPC) and ischemic preconditioning (IPC) require activation of sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase, to induce neuroprotection against cerebral ischemia. In the present study, we tested two hypotheses: (a) that neuroprotection against cerebral ischemia can be induced by RPC in vivo; and (b) that RPC neuroprotection involves alterations in mitochondrial function via the SIRT1 target mitochondrial uncoupling protein 2 (UCP2). IPC was induced by 2 min of global ischemia (temporary bilateral carotid artery occlusion with hypotension), and RPC, by i.p. injection of resveratrol at 10, 50 and 100 mg/kg dosages. Forty-eight hours later, we compared the neuroprotective efficacy of RPC and IPC in vulnerable cornu ammonis 1 hippocampal pyramidal neurons using a rat model of asphyxial cardiac arrest (ACA). SIRT1 activity was measured using a SIRT1-specific fluorescent enzyme activity assay. In hippocampal mitochondria isolated 48 h after IPC or RPC, we measured UCP2 levels, membrane potential, respiration, and the mitochondrial ATP synthesis efficiency (ADP/O ratio). Both IPC and RPC induced tolerance against brain injury induced by cardiac arrest in this in vivo model. IPC increased SIRT1 activity at 48 h, while RPC increased SIRT1 activity at 1 h but not 48 h after treatment in hippocampus. Resveratrol significantly decreased UCP2 levels by 35% compared to sham-treated rats. The SIRT1-specific inhibitor sirtinol abolished the neuroprotection afforded by RPC and the decrease in UCP2 levels. Finally, RPC significantly increased the ADP/O ratio in hippocampal mitochondria reflecting enhanced ATP synthesis efficiency. In conclusion, in vivo resveratrol pretreatment confers neuroprotection similar to IPC via the SIRT1–UCP2 pathway.