NADH as a functional imaging signal
Karl A. Kasischke
Dept. of Neurosurgery, Center for Aging and Developmental Biology, University of Rochester Medical Center, USA
Nicotinamide adenine dinucleotide (NADH) is the principle electron carrier in glycolytic and oxidative metabolism. Importantly, the reduced co-enzyme (NADH) is fluorescent, while the oxidized co-enzyme (NAD +) is not. Therefore, intrinsic NADH fluorescence gives a direct measure of the cellular NADH/NAD +-ratio and has been utilized as an indicator for both oxidative (Chance&Williams, 1955; Chance et al., 1962) and glycolytic (Aubert et al., 1964; Williamson et al., 1967) metabolism. In brain, the tissue concentration of nicotinamide adenine dinucleotide phosphate (NADPH), the other fluorescent pyridine nucleotide, is minor compared to NADH.
The principle of two-photon NADH imaging is that a native intracellular molecule present in millimolar concentrations is visualized in live animals or tissues using non-linear laser-scanning microscopies. Advantages over established functional imaging modalities are the absence of dyes and tracers, spatial resolution ranging from the columnar to the subcellular level, and temporal resolution below the response functions of glycolytic or oxidative metabolism and cerebral blood flow. In addition, because of its unique molecular structure and its specific binding to cytoplasmic and mitochondrial dehydrogenases, NADH is a probe of the cellular microenvironment readily accessible to steady-state and time-resolved spectroscopy and possibly imaging spectroscopy.
Using two-photon NADH imaging we have resolved activity-dependent oxidative and glycolytic metabolic signatures in processes of astrocytes and neurons in brain tissue-slices. Based on these observations we have proposed a unifying model for neurometabolic coupling, in which early oxidative metabolism in neurons is eventually sustained by late activation of glycolysis in astrocytes (Kasischke et al., 2004). Recently, we have extended two-photon NADH imaging into time-resolved NADH spectroscopy (Vishwasrao et al., 2005) with the resolution of free/bound states and conformational changes of intracellular NADH. This allows for a largely improved quantification of NADH fluctuations and ultimately lays the foundation for quantitative imaging of neural energy metabolism. Latest results (Kasischke&Nedergaard, unpublished) show that two-photon NADH imaging in-vivo gives a precise representation of biochemical disturbances in the mouse cortex upon hypoxemia which appear to be highly dependent on their location within the capillary bed.