Abstract
Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.
Highlights
Healthy brain function consists of intricate, relentless electrochemical signaling between neurons, astrocytes, and cerebral microvasculature, and these energetically-demanding processes rely critically upon oxidative metabolism and a tightly-regulated supply of metabolites such as oxygen and glucose [1]
We previously demonstrated in vivo 2-photon microscopy (2PM)-based fluorescence lifetime imaging (FLIM) (2P-FLIM) measurements of cerebral NADH in anesthetized rats, and observed how cerebral NADH fluorescence can be resolved into 4 distinct lifetime components whose amplitudes change rapidly with anoxia and recovery [19]
Fluorescence signal from astrocytes labelled with Sulphorhodamine 101 (SR101) dye was detected in a second channel, providing morphological information and ensuring that identical fields of view were imaged before and after metabolic manipulation (Figs. 2(b)-2(e))
Summary
Healthy brain function consists of intricate, relentless electrochemical signaling between neurons, astrocytes, and cerebral microvasculature, and these energetically-demanding processes rely critically upon oxidative metabolism and a tightly-regulated supply of metabolites such as oxygen and glucose [1]. We previously demonstrated in vivo 2PM-based FLIM (2P-FLIM) measurements of cerebral NADH in anesthetized rats, and observed how cerebral NADH fluorescence can be resolved into 4 distinct lifetime components whose amplitudes change rapidly with anoxia and recovery [19]. These reports suggest that lifetime-based analysis of NADH fluorescence shows great promise for distinguishing variations in metabolism with much higher specificity than more conventional intensity-based measurements of NADH fluorescence. A detailed understanding of the underlying biochemical significance of FLIM-based observations of NADH is currently lacking and limits its rigorous interpretation
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