Mitochondrial regulation of ishcemic neuronal death

我们已经完成了两个项目的支持，这笔赠款。首先，我们研究了OGD（氧-糖剥夺）后线粒体膜电位与死亡方式、凋亡和坏死的关系。其次，我们研究了氧-糖剥夺后再氧合时线粒体膜电位。第一个项目提出了再灌注早期神经元对自由基毒性作用的生理保护机制。因此，我们转移到线粒体机制参与主动神经元自杀，凋亡，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的关系我们在神经元细胞培养中采用氧葡萄糖耗竭（OGD）来复制缺血条件，并检测MMP，OGD期间高能磷酸盐的消耗和随后的死亡模式。MMP超极化（标准化JC-1 f 关于我们 MMP在30分钟OGD后30分钟复氧时变为去荧光（标准化JC-1荧光0.58 +/- 0.10）。600 GD后MMP的结果变化（标准化JC-1荧光1.12 +/-0.13）。在30 min OGD后3 h，大多数神经元存活（76%），而在90 min OGD后3 h，大多数神经元死亡（72%）。在30 min OGD后24 h，大多数神经元（66%）为TUNEL阳性。OGD 90 min后仅有少数神经元（12%）TUNEL阳性。细胞色素c在30分钟OGD和90分钟OGD后复氧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。缺氧30 min和缺氧30 min后复氧30 min时细胞内ATP含量分别为8.1±6.6%和3.2±1.9%，缺氧60 min对ATP含量无明显影响（7.1± 5.8%和2.6±0.5%）。长OGD导致去极化，而短OGD诱导超极化。OGD后的超极化并不伴随ATP的产生。这一观察结果表明，在复氧和FoF 1-ATP合酶的干扰电子再进入内膜的抑制。神经元在超极化过程中仍然存活，但这种超极化似乎与随后的凋亡表现有关。线粒体膜电位的耗散似乎是严重能量缺乏的结果，并导致坏死。线粒体的超极化反映了ATP产生的紊乱，可能是一个启动凋亡级联反应的过程。2.异丙酚对缺氧缺糖后ATP消耗及随后神经元死亡的影响原代培养的海马细胞缺氧缺糖30 min（30 OGD）或90 min（90 OGD）。以0.1μM（Pro0.1）或1.0μM（Pro1.0）的浓度向培养物中加入丙泊酚。使用荧光素酶-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含量降低（0.276±0.115μM/μg（对照），0.172±0.125μM/μg（30 OGD），0.096±0.092μM/μg（90 OGD））。异丙酚不改变ATP含量。30 OGD后MMP超极化（1.26±0.23（溶剂）、1.29±0.13（Pro0.1）和1.18±0.06（Pro1.0）），但90 OGD后接近去极化（0.77±0.04（溶剂）、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%（溶剂）、88.6±4.6%（Pro0.1）和84.5±4.1%（Pro1.0）降至90 OGD后的31.7±18.5%（溶剂）、43.6±25.0%（Pro0.1）和56.9±20.2%（Pro1.0）。在OGD后24 h，30 OGD组中TUNEL阳性细胞增加至34.5±6.2%（溶剂）、26.7±7.9%（Pro0.1）和30.4±7.1%（Pro1.0）。异丙酚对细胞凋亡的发生率没有药理学作用，异丙酚抑制急性神经元死亡，但不能阻止细胞过度穿孔和随后的细胞凋亡。异丙酚诱导神经元死亡暂停，在此期间，药物干预可能能够防止细胞死亡。少

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