Ward Manus W, Huber Heinrich J, Weisová Petronela, Düssmann Heiko, Nicholls David G, Prehn Jochen H M
Department of Physiology and Medical Physics and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
J Neurosci. 2007 Aug 1;27(31):8238-49. doi: 10.1523/JNEUROSCI.1984-07.2007.
A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of delta psi(m) that was closely associated with a loss of delta psi(p) and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.
线粒体生物能量学的功能障碍已被证明与凋亡性和坏死性神经元损伤的发生密切相关。在此，我们开发了一种自动化计算模型，该模型将四甲基罗丹明甲酯（TMRM）的单细胞荧光解释为线粒体膜电位（Δψm）或质膜电位（Δψp）变化的结果，从而能够对单细胞群体的反应进行表征并进行后续统计分析。长时间谷氨酸兴奋引发的坏死性损伤导致Δψm迅速单相或双相丧失，这与Δψp的丧失以及神经元烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和三磷酸腺苷（ATP）水平的快速下降密切相关。短暂谷氨酸兴奋诱导的延迟性凋亡损伤导致TMRM荧光出现小幅、可逆的下降，随后使用对Δψp敏感的阴离子探针二苯甲酰甲烷（DiBAC2(3)）证实Δψm持续超极化。Δψm的这种超极化与神经元葡萄糖摄取、NADPH可用性和ATP水平的显著增加密切相关。在单细胞水平对Δψm或Δψp变化的统计分析揭示了两个主要相关性；那些在谷氨酸兴奋初始阶段显示出更明显的Δψp去极化的神经元更快进入凋亡，而在谷氨酸兴奋后显示出更明显的Δψm超极化的神经元存活时间更长。事实上，那些对短暂谷氨酸兴奋耐受的神经元（18%）显示出Δψm的最显著增加。我们的结果表明Δψm的超极化与兴奋性毒性损伤期间葡萄糖摄取增加、NADPH可用性增加和存活反应相关。
