Mitochondrial regulation of ishcemic neuronal death

缺血性神经元死亡的线粒体调节

• 批准号: 15591658
• 负责人: IIJIMA Takehiko
• 金额: $ 2.18万
• 依托单位: Kyorin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591658/
• 关键词: cerebral ischemia; mitochondria; membrane potential; apoptosis; necrosis; ATP; Ca deregultaion; TUNEL method; mitochondria; neuronal death; hyperpolarization; membrane potential; Apoptosis; ischemia; アポトーシス; ミトコンドリア; 脳虚血; 培養神経細胞; OGD; 膜

We have accomplished two projects supported by this grant. First, we examined the relationship between mitochondrial membrane potential after OGD (oxygen-glucose deprivation) and death modes, apoptosis and necrosis Second, we examined mitochondrial membrane potential during reoxygenation after oxygen-glucose deprivation. The first project suggested physiological protective mechanism of neuron from toxic effect of free radical during early reperfusion phase. Therefore, we moved to mitochondrial mechanism involved in active neuronal suicide, apoptosis because active neuronal death executed by neuron itself seems to be a main cause of neuronal damage after ischemia.1.The relationship between mitochondrial membrane potential and death modesWe employed oxygen glucose depletion (OGD) in neuronal cell culture to reproduce ischemic condition and examined the relationship between MMP, consumption of high-energy phosphate and subsequent death mode during OGD.MMP hyperpolarized (normalized JC-1 f … More luorescence 1.98 +/- 0.11, mean +/- SD, control=1.0) at 30 min reoxygenation following 30min OGD, while MMP turned to depolarize (normalized JC-1 fluorescence 0.58 +/- 0.10) at 30 min reoxygenation following 90min OGD. The result of MMP following 600GD varied (normalized JC-1 fluorescence 1.12 +/- 0.13). Most neurons were viable (76%) at 3 hrs following 30min OGD, while most neuron was dead (72%) at 3 hrs following 90 min OGD. Most neurons (66%) were TUNEL positive at 24 hours following 30 min OGD. Only a few neurons (12%) were TUNEL positive following 90 min OGD. Cytochrome c became diffuse in cytoplasma at 24 hrs of reoxygenation following 30 min OGD and 90 min OGD, and was already diffuse even at 3 hrs following 90 min OGD. The intracellular ATP content was 8.1±6.6% and 3.2±1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively ; 60 min OGD did not significantly change these levels (7.1±5.8%, 2.6±0.5%).Mitochondria membrane potential during reoxygenation following OGD depends on the length of OGD. Long OGD results in depolarization, while shorter OGD induces hyperpolarization. Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. Neurons are still viable during hyperpolarization, but this hyperpolarization seems to link the subsequent manifestation of apoptosis. The dissipation of mitochondrial membrane potential seems to be a consequence of severe energy deficit and results in necrosis. Hyperpolarization of mitochondria seems to reflect disturbance of ATP production, and may be a process to switch on apoptotic cascade.2.The effect of propofol on consumption of ATP after OGD and subsequent neuronal deathPrimary hippocampal cell culture was incubated with oxygen-glucose deprivation for 30 min (30OGD) or 90 min (90OGD). Propofol was added to the culture at a concentration of 0.1μM (Pro0.1) or 1.0μM (Pro1.0). ATP content was assayed using the luciferin-luciferase reaction. Neuronal viability and appearance of apoptosis was assessed. ATP content was decreased after OGD (0.276±0.115μM/μg (control), 0.172±0.125μM/μg (30OGD), and 0.096±0.092μM/μg (90OGD)). Propofol did not alter ATP content. MMP was hyperpolarized after 30OGD (1.26±0.23 (vehicle), 1.29±0.13 (Pro0.1), and 1.18±0.06 (Pro1.0)) but approached depolarization after 90OGD (0.77±0.04 (vehicle), 0.89±0.04 (Pro0.1), and 1.03±0.15 (Pro1.0)). Viability of cells decreased from 91.8±2.0% (vehicle), 88.6±4.6% (Pro0.1), and 84.5±4.1% (Pro1.0) in control conditions to 31.7±18.5% (vehicle), 43.6±25.0% (Pro0.1), and 56.9±20.2% (Pro1.0) after 90OGD. At 24 h after OGD, TUNEL-positive cells were increased to 34.5±6.2% (vehicle), 26.7±7.9% (Pro0.1), and 30.4±7.1% (Pro1.0) in the 30OGD group. No pharmacological effect of propofol on the incidence of apoptosis was found. Propofol inhibited acute neuronal death, but did not prevent hyperporalization and subsequent apoptosis. Propofol induces a moratorium on neuronal death, during which pharmacological intervention might be able to prevent cell death. Less

我们已经完成了这笔赠款支持的两个项目。首先，我们检查了OGD（氧糖剥夺）后线粒体膜电位与死亡模式、细胞凋亡和坏死之间的关系。其次，我们检查了氧糖剥夺后复氧过程中的线粒体膜电位。第一个项目提出了神经元在再灌注早期免受自由基毒性作用的生理保护机制。因此，我们转向参与主动神经元自杀、凋亡的线粒体机制，因为神经元本身执行的主动神经元死亡似乎是缺血后神经元损伤的主要原因。 1.线粒体膜电位与死亡模式之间的关系我们在神经细胞培养物中采用氧糖耗竭（OGD）来重现缺血状态，并检查了线粒体膜电位与死亡模式之间的关系。 MMP、OGD 期间高能磷酸盐的消耗和随后的死亡模式。在 30 分钟 OGD 后 30 分钟复氧时，MMP 超极化（归一化 JC-1 f ... More 荧光 1.98 +/- 0.11，平均值 +/- SD，对照=1.0），而在复氧后 30 分钟后，MMP 转为去极化（归一化 JC-1 荧光 0.58 +/- 0.10） 90 分钟 OGD。 600GD 后的 MMP 结果有所不同（标准化 JC-1 荧光 1.12 +/- 0.13）。大多数神经元在 30 分钟 OGD 后 3 小时内仍存活 (76%)，而大多数神经元在 90 分钟 OGD 后 3 小时内死亡 (72%)。大多数神经元 (66%) 在 30 分钟 OGD 后 24 小时呈 TUNEL 阳性。 90 分钟 OGD 后，只有少数神经元 (12%) 呈 TUNEL 阳性。细胞色素c在30分钟OGD和90分钟OGD后的再氧合24小时时在细胞质中扩散，并且甚至在90分钟OGD后3小时时已经扩散。 30分钟OGD后和30分钟OGD后30分钟复氧后，细胞内ATP含量分别为8.1±6.6%和3.2±1.9%； 60 分钟 OGD 没有显着改变这些水平 (7.1±5.8%, 2.6±0.5%)。 OGD 后复氧期间的线粒体膜电位取决于 OGD 的长度。较长的 OGD 会导致去极化，而较短的 OGD 则会引起超极化。 OGD 后的超极化并不伴随 ATP 的产生。这一观察结果表明，在再氧合过程中电子重新进入内膜受到抑制，并且 FoF1-ATP 合酶受到干扰。神经元在超极化期间仍然存活，但这种超极化似乎与随后的细胞凋亡表现有关。线粒体膜电位的耗散似乎是严重能量不足的结果并导致坏死。线粒体的超极化似乎反映了ATP产生的紊乱，并且可能是启动凋亡级联的过程。2.异丙酚对OGD后ATP消耗和随后的神经元死亡的影响原代海马细胞培养物缺氧葡萄糖培养30分钟（30OGD）或90分钟（90OGD）。将异丙酚以 0.1μM (Pro0.1) 或 1.0μM (Pro1.0) 的浓度添加到培养物中。使用荧光素-荧光素酶反应测定 ATP 含量。评估神经元活力和细胞凋亡的出现。 OGD后ATP含量下降（0.276±0.115μM/μg（对照）、0.172±0.125μM/μg（30OGD）和0.096±0.092μM/μg（90OGD））。异丙酚不会改变 ATP 含量。 MMP 在 30OGD 后超极化（1.26±0.23（车辆）、1.29±0.13（Pro0.1）和 1.18±0.06（Pro1.0）），但在 90OGD 后接近去极化（0.77±0.04（车辆）、0.89±0.04（Pro0.1）和1.03±0.15（Pro1.0））。细胞活力从对照条件下的 91.8±2.0% (vehicle)、88.6±4.6% (Pro0.1) 和 84.5±4.1% (Pro1.0) 下降至 31.7±18.5% (vehicle)、43.6±25.0% (Pro0.1) 和 56.9±20.2% (Pro1.0)。 90元。 OGD后24小时，30OGD组中TUNEL阳性细胞增加至34.5±6.2%（载体）、26.7±7.9%（Pro0.1）和30.4±7.1%（Pro1.0）。未发现异丙酚对细胞凋亡发生率有药理作用。异丙酚抑制急性神经元死亡，但不能阻止超多孔化和随后的细胞凋亡。异丙酚可延缓神经元死亡，在此期间药物干预可能能够防止细胞死亡。较少的

项目成果

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

• DOI: 10.1016/s0197-0186(02)00228-0
• 发表时间: 2003-08-01
• 期刊: NEUROCHEMISTRY INTERNATIONAL
• 影响因子: 4.2
• 作者: Iijima, T;Mishima, T;Iwao, Y
• 通讯作者: Iwao, Y

Mitochondrial membrane potential, its relation to intracellular ATP and subsequent death mode. accepted for publication

线粒体膜电位，其与细胞内 ATP 和随后死亡模式的关系。

• 期刊: Focus in Neurochemistry Research Nova Science Publishers.Inc.NY USA (in press)
• 作者: Iijima T;Mishima;T.;Akagawa;K.;Iwao;Y.;Iijima T
• 通讯作者: Iijima T

Iijima T, Mishima T, Akagawa K, Iwao Y: "Mitochondrial hyperpolarization after transient oxygen-glucose deprivation and subsequent apoptosis in cultured rat hippocampal neurons"Brain Research. 993. 140-145 (2003)

Iijima T、Mishima T、Akakawa K、Iwao Y：“培养的大鼠海马神经元短暂氧葡萄糖剥夺后线粒体超极化和随后的细胞凋亡”脑研究。

Iijima T, Mishima T, Akagawa K, Iwao Y: "Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron"Neurochemistry International. 43(3). 263-269 (2003)

Iijima T、Mishima T、Akakawa K、Iwao Y：“培养的海马神经元短暂实验性缺血后的线粒体膜电位和细胞内 ATP 含量”《神经化学国际》。

Mitochondrial hyperpolarization after transient oxygen-glucose deprivation and subsequent apoptosis in cultured rat hippocampal neurons

• DOI: 10.1016/j.brainres.2003.09.041
• 发表时间: 2003-12-12
• 期刊: BRAIN RESEARCH
• 影响因子: 2.9
• 作者: Iijima, T;Mishima, T;Iwao, Y
• 通讯作者: Iwao, Y