Tufts University, Department of Biomedical Engineering, Medford, MA, 02155, USA.
University of Arkansas, Department of Biomedical Engineering, Fayetteville, AR, 72701, USA.
Sci Rep. 2017 Apr 21;7(1):1041. doi: 10.1038/s41598-017-01015-9.
As neurodegenerative conditions are increasingly linked to mitochondrial dysfunction, methods for studying brain cell metabolism at high spatial resolution are needed to elucidate neurodegeneration mechanisms. Two-photon excited fluorescence (TPEF) imaging is a non-destructive, high-resolution technique for studying cell metabolism via endogenous fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). We employed TPEF to study the metabolism of primary rat astrocyte and neuronal cultures under normal growth conditions and in response to manganese (Mn) treatment. Histograms of pixel-wise optical redox ratio, defined as FAD/(FAD + NAD(P)H), revealed three distinct redox distributions and significant differences in their relative weights between astrocytes and neurons. When treated with Mn, both cell types exhibited redox ratio shifts consistent with increased oxidative stress. However, the manner in which the redox distributions was affected was distinct for the two cell types. Furthermore, NAD(P)H fluorescence lifetime imaging revealed an increase in bound NAD(P)H fraction upon Mn treatment for neurons, consistent with enhanced apoptosis. Astrocytes showed a decrease in bound fraction, possibly due to a shift towards glycolytic metabolism in response to impaired respiration. These results exhibit TPEF's utility for characterizing detailed metabolic changes of different brain cell types in response to neurotoxins.
随着神经退行性疾病与线粒体功能障碍的关联日益密切，需要能够以高空间分辨率研究脑细胞代谢的方法来阐明神经退行性变机制。双光子激发荧光（TPEF）成像技术是一种非破坏性的、高分辨率技术，可通过还原型烟酰胺腺嘌呤二核苷酸（磷酸）（NAD(P)H）和黄素腺嘌呤二核苷酸（FAD）的内源性荧光来研究细胞代谢。我们利用 TPEF 研究了在正常生长条件下以及在锰（Mn）处理下原代大鼠星形胶质细胞和神经元培养物的代谢。像素级光氧化还原比的直方图定义为 FAD/（FAD+NAD(P)H），揭示了星形胶质细胞和神经元之间存在三种不同的氧化还原分布，并且它们的相对权重存在显著差异。用 Mn 处理后，两种细胞类型的氧化还原比均发生了变化，表明氧化应激增加。然而，两种细胞类型的氧化还原分布受影响的方式不同。此外，NAD(P)H 荧光寿命成像显示神经元中结合型 NAD(P)H 分数增加，这与增强的细胞凋亡一致。星形胶质细胞中结合分数降低，可能是由于呼吸受损导致糖酵解代谢增强所致。这些结果表明 TPEF 可用于表征不同脑细胞类型对神经毒素的详细代谢变化。
