Vascular and metabolic dysfunctions are well known features of Alzheimer’s disease (AD) and they precede clinical dementia. Undoubtedly vascular changes are expected as amyloid accumulates in the arterial vessel walls in cerebral amyloid angiopathy (CAA), leading to the death of smooth muscle cells, cerebral hypoperfusion and inadequate oxygen supply. These vascular events could also contribute to metabolic alterations in glucose homeostasis. High resolution in vivo study of the dynamic vascular and metabolic events may reveal which tissue regions and cell populations are affected and cast light on the mechanisms that contribute to AD pathogenesis.


      We used fluorescence imaging of nicotinamide adenine dinucleotide (NADH) as an intrinsic marker for cellular metabolic states and tissue oxygen supply in vivo. We resolved the tissue boundaries of NADH fluorescence in the cortex of transgenic AD mice (B6C3.Tg(APPswe-PSEN1de9), n=4, 12-24 months old) and observed NADH pattern relative to vessels during hyperoxia and normoxia. We then used in vivo two-photon fluorescence microscopy together with cell-type specific labeling to determine the cellular origin of the intrinsic signal and the locality of CAA.


      Reduction of oxygen supply from hyperoxia to normoxia produced no detectable changes in controls, however AD mice showed characteristic NADH pattern (Figure 1A), indicative of reduced oxygen gradient and rise in glycolysis in tissues further away from the arterial oxygen supply. Areas around capillary beds showed decreased NADH signal. Two-photon imaging under the same conditions revealed numerous cells with increased signal (Figure 1B) and only some of those cells stained positive for the astrocyte marker Sulforhodamine-101 (Figure 1C). All AD mice had CAA and tissue plaques seen with Methoxy-X04 staining (Figure 1D) and there appeared to be no association of the NADH signal with the plaques location.


      In agreement with previous findings, double transgenic AD mice display chronic tissue hypoxia. Our preliminary results also indicate that under those conditions a subset of cells may adapt by up-regulating glycolysis to overcome the deficient oxidative phosphorylation. The population of cells with increased NADH signal is likely a combination of neurons and glia. This work can lead to new strategies that target metabolic pathways to halt AD progression.
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      Figure 1In vivo NADH fluorescence imaging of AD mice reveals chronic tissue hypoxia and cellular metabolic shift. A) Characteristic NADH pattern after reduction of oxygen supply. Arrowheads pointing to a vein; asterisks at arteries. B) Two-photon imaging under the same conditions shows numerous cells with increased signal (green). C) A subset of those cells stain positive for the astrocyte marker SR101 (red). Arrowheads at cells double positive for NADH and SR101. D) Cortex also shows heavy CAA (blue) and tissue plaques (blue, asterisks).