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.
Keywords: fiber-optic probes; fluorescent protein sensors; neurophotonics; optogenetics.
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