Physics Department, M.V. Lomonosov Moscow State University, Moscow, Russia.
M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
J Biophotonics. 2022 Oct;15(10):e202200050. doi: 10.1002/jbio.202200050. Epub 2022 Jul 8.
We present an experimental framework and methodology for in vivo studies on rat stroke models that enable a real-time fiber-optic recording of stroke-induced hydrogen peroxide and pH transients in ischemia-affected brain areas. Arrays of reconnectable implantable fiber probes combined with advanced optogenetic fluorescent protein sensors are shown to enable a quantitative multisite time-resolved study of oxidative-stress and acidosis buildup dynamics as the key markers, correlates and possible drivers of ischemic stroke. The fiber probes designed for this work provide a wavelength-multiplex forward-propagation channel for a spatially localized, dual-pathway excitation of genetically encoded fluorescence-protein sensors along with a back-propagation channel for the fluorescence return from optically driven fluorescence sensors. We show that the spectral analysis of the fiber-probe-collected fluorescence return provides means for a high-fidelity autofluorescence background subtraction, thus enhancing the sensitivity of real-time detection of stroke-induced transients and significantly reducing measurement uncertainties in in vivo acute-stroke studies as inherently statistical experiments operating with outcomes of multiply repeated measurements on large populations of individually variable animal stroke models.
我们提出了一种用于大鼠中风模型的体内研究的实验框架和方法，该方法能够实时记录中风引起的过氧化物氢和 pH 瞬变在受影响的大脑区域。可重新连接的植入式光纤探头阵列与先进的光遗传学荧光蛋白传感器相结合，可实现对氧化应激和酸中毒形成动力学的定量多部位时间分辨研究，作为缺血性中风的关键标志物、相关物和可能的驱动因素。为这项工作设计的光纤探头提供了一个波长多路复用前向传播通道，用于沿遗传编码荧光蛋白传感器进行空间局部、双通道激发，以及用于从光驱动荧光传感器返回的荧光的后向传播通道。我们表明，对光纤探头收集的荧光返回的光谱分析提供了一种高保真度的自发荧光背景扣除手段，从而提高了对中风诱导瞬变的实时检测的灵敏度，并在体内急性中风研究中显著降低了测量不确定性，因为这些研究作为具有大量个体变量动物中风模型的多次重复测量结果的固有统计实验来操作。
