Investigating cerebral metabolism in vivo at a microscopic level is essential for understanding brain function and its pathological alterations. The intricate signaling and metabolic dynamics between neurons, glia, and microvasculature requires much more detailed understanding to better comprehend the mechanisms governing brain function and its disease-related changes. We recently demonstrated that pharmacologically-induced alterations to different steps of cerebral metabolism can be distinguished utilizing 2-photon fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence in vivo. Here, we evaluate the ability of the phasor analysis method to identify these pharmacological metabolic alterations and compare the method's performance with more conventional nonlinear curve-fitting analysis. Visualization of phasor data, both at the fundamental laser repetition frequency and its second harmonic, enables resolution of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism. Compared to our previous classification models based on nonlinear curve-fitting, phasor-based models required fewer parameters and yielded comparable or improved classification accuracy. Fluorescence lifetime imaging of NADH and phasor analysis shows utility for detecting metabolic alterations and will lead to a deeper understanding of cerebral energetics and its pathological changes.

• MeSH
• bikukulin analogy a deriváty   farmakologie   MeSH
• biologické markery metabolismus   MeSH
• biologické modely MeSH
• hlodavci fyziologie   MeSH
• intravitální mikroskopie metody   MeSH
• lidé MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• mozková kůra účinky léků   metabolismus   MeSH
• NAD metabolismus   MeSH
• nelineární dynamika MeSH
• potkani Sprague-Dawley MeSH
• záchvaty chemicky indukované   diagnostické zobrazování   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• bicuculline methiodide MeSH Prohlížeč
• bikukulin MeSH
• biologické markery MeSH
• NAD MeSH

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.

• Klíčová slova
• (170.0170) Medical optics and biotechnology, (170.3650) Lifetime-based sensing, (180.4315) Nonlinear microscopy,
• Publikační typ
• časopisecké články MeSH