Mitochondrial regulation of ishcemic neuronal death

我们已经完成了这一资助下的两个项目。首先，我们研究了OGD（氧-葡萄糖剥夺）后线粒体膜电位与死亡模式、细胞凋亡和坏死的关系；其次，我们研究了氧-葡萄糖剥夺后再氧化过程中的线粒体膜电位。第一个项目提出了再灌注早期自由基对神经元毒性的生理保护机制。因此，我们转向线粒体机制参与神经元的活动性自杀、凋亡，因为神经元自身执行的活动性神经元死亡似乎是神经元缺血后损伤的主要原因。线粒体膜电位与死亡模式的关系我们采用氧葡萄糖耗竭（OGD）在神经元细胞培养中再现缺血状态，并研究了在OGD期间MMP、高能磷酸盐消耗与随后死亡模式之间的关系。在OGD 30min后30min再氧化时，MMP超极化（归一化JC-1荧光1.98 +/- 0.11，平均+/- SD，对照=1.0），而在OGD 90min后30min再氧化时，MMP转为去极化（归一化JC-1荧光0.58 +/- 0.10）。600GD后MMP结果变化（归一化JC-1荧光1.12 +/- 0.13）。大多数神经元在30min后3小时存活（76%），而大多数神经元在90min后3小时死亡（72%）。大多数神经元（66%）在OGD 30min后24小时TUNEL阳性。OGD 90min后，只有少数神经元（12%）呈TUNEL阳性。细胞色素c在OGD 30min和OGD 90min后再氧化24小时时在胞浆中弥漫性扩散，甚至在OGD 90min后3小时就已经弥漫性扩散。OGD 30min后细胞内ATP含量为8.1±6.6%，OGD 30min后再氧30min后细胞内ATP含量为3.2±1.9%；60分钟OGD没有显著改变这些水平（7.1±5.8%,2.6±0.5%）。OGD后再氧化过程中的线粒体膜电位取决于OGD的长度。长OGD导致去极化，短OGD导致超极化。OGD后的超极化不伴随ATP的产生。这一观察结果表明，在再氧化过程中，电子重新进入内膜的抑制和FoF1-ATP合成酶的干扰。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产生的干扰，可能是凋亡级联反应的一个过程。异丙酚对OGD后ATP消耗及随后神经元死亡的影响原代海马细胞培养在缺氧-葡萄糖剥夺条件下培养30min （30OGD）或90min （90OGD）。在培养液中加入浓度为0.1μM （Pro0.1）或1.0μM （Pro1.0）的异丙酚。采用荧光素-荧光素酶反应测定ATP含量。观察神经元活力和细胞凋亡的表现。OGD处理后ATP含量分别为0.276±0.115μM/μg（对照组）、0.172±0.125μM/μg （30OGD）和0.096±0.092μM/μg （90OGD）。异丙酚没有改变ATP的含量。MMP在30OGD后呈超极化（1.26±0.23 （vehicle）、1.29±0.13 （Pro0.1）和1.18±0.06 (Pro1.0)），而在90OGD后呈去极化（0.77±0.04 （vehicle）、0.89±0.04 （Pro0.1）和1.03±0.15 (Pro1.0)）。细胞活力从对照组的91.8±2.0%（载体）、88.6±4.6% （Pro0.1）和84.5±4.1% （Pro1.0）下降到90OGD后的31.7±18.5%（载体）、43.6±25.0% （Pro0.1）和56.9±20.2% （Pro1.0）。OGD后24 h， 30OGD组tunel阳性细胞比例分别为34.5±6.2%（对照）、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，但不阻止过度极化和随后的细胞凋亡。异丙酚诱导暂停神经元死亡，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