Metabolism and oxygen tension in Alzheimer's disease mouse model using two-photon FLIM-PLIM microscopy

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and memory deficits, processes critically linked to mitochondrial dysfunction in the nervous system. Equally, adequate brain tissue oxygenation is essential for cerebral metabolism, and its impairment further compromises neuronal energy homeostasis. Together, mitochondrial dysfunction and disrupted cerebral oxygenation represent key interconnected pathological mechanisms underlying AD progression, highlighting the significance of targeting these pathways in understanding and managing the disease.

Cerebral energy metabolism in the cortex of wild-type (WT) and Alzheimer's disease (AD) model mice was investigated using minimally invasive two-photon FLIM, with the protein-bound fraction of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) a2% serving as a metric for mitochondrial metabolic activity. The probe " $\mathrm{Ru}(\mathrm{BPY}{)}_3$ " was calibrated to examine the dynamic nature of its phosphorescence lifetime to estimate the partial pressure of oxygen ( ${\mathrm{pO}}_2$ ) in brain vasculature of live animals.

Simultaneous two-photon fluorescence and phosphorescence lifetime imaging (2P-FLIM-PLIM) provides a minimally invasive approach to measure the metabolic state in combination with the ${\mathrm{pO}}_2$ in the vascularity of live animals. The metabolic state is obtained via FLIM of NAD(P)H, ${\mathrm{pO}}_2$ via the phosphorescence lifetime of $\mathrm{Ru}(\mathrm{BPY}{)}_3$ , an oxygen-sensitive exogenous probe.

AD mouse cortices exhibit higher level of NAD(P)H a2% and lower level of ${\mathrm{pO}}_2$ compared with WT at anesthetized resting-state. 2P-PLIM investigation reveals that the lower level of ${\mathrm{pO}}_2$ in vascular network Alzheimer's disease (AD) mice cortex indicates increased oxygen consumption in AD.

The concurrent application of simultaneous 2P-FLIM-PLIM in the mouse brain establishes this dual-modality protocol for the simultaneous assessment of cerebral metabolic alterations using biomarker NAD(P)H and ruthenium-based cerebrovascular oxygen tension measurements. These findings underscore the utility of this multiparametric optical imaging strategy in elucidating the interplay between mitochondrial metabolic dysfunction and impaired brain tissue oxygenation, providing a compelling platform for advancing mechanistic investigations into the neuropathological sequelae of Alzheimer's disease.