In Vivo Imaging of Flavoprotein Fluorescence During Hypoxia Reveals the Importance of Direct Arterial Oxygen Supply to Cerebral Cortex Tissue

Abstract

Live imaging of mitochondrial function is crucial to understand the important role played by these organelles in a wide range of diseases. The mitochondrial redox potential is a particularly informative measure of mitochondrial function, and can be monitored using the endogenous green fluorescence of oxidized mitochondrial flavoproteins. Here, we have observed flavoprotein fluorescence in the exposed murine cerebral cortex in vivo using confocal imaging; the mitochondrial origin of the signal was confirmed using agents known to manipulate mitochondrial redox potential. The effects of cerebral oxygenation on flavoprotein fluorescence were determined by manipulating the inspired oxygen concentration. We report that flavoprotein fluorescence is sensitive to reductions in cortical oxygenation, such that reductions in inspired oxygen resulted in loss of flavoprotein fluorescence with the exception of a preserved 'halo' of signal in periarterial regions. The findings are consistent with reports that arteries play an important role in supplying oxygen directly to tissue in the cerebral cortex, maintaining mitochondrial function.

Keywords: Brain; Confocal microscope; Mitochondria; Oxygen; Vasculature.

Fig. 29.1

(a) Fluorescence intensity in response to NaCN and FCCP. Scale bar = 100 μm. (b) Quantification of fluorescence intensity before and after application of saline/DMSO or NaCN/FCCP to the cortex. Data are normalised to signal intensity before treatment and displayed as mean ± SEM. Statistical significance was assessed using a paired sample t-test (*p ≤ 0.05, **p ≤ 0.01)

Fig. 29.2

(a) Flavoprotein (green) and TMRM (red) fluorescence in response to changes in FiO₂. Scale bar = 200 μm. (b) The ratio of periarterial to perivenular tissue flavoprotein fluorescence intensity (examples indicated in a), red  = periarterial and blue = perivenular. Data are displayed as mean ± SEM. Statistical significance was assessed using a paired sample t-test (*p ≤ 0.05, ***p ≤ 0.001)

Fig. 29.3

(a) Flavoprotein (green) and NAD(P)H (blue) fluorescence in response to hypoxia. (b) Oxygen-sensitive phosphorescent beads (red) on the hypoxic cortex. A = Periarterial, NA = Nonarterial. (c) Bead emission intensity in response to changes in FiO₂. Data are displayed as mean ± SEM. Statistical significance was assessed using an independent sample t-test (**p≤0.01)