Cortical NADH during pharmacological manipulations of the respiratory chain and spreading depression in vivo

Abstract

The nicotinamide adenine dinucleotide (NADH) is one of the main means for energy transfer in the mitochondrial respiratory chain and is an important parameter of cellular metabolism. NADH can be measured by its fluorescence and various fluorometric methods have been developed. In this study, a pulsed nitrogen laser combined with a fibreoptic set-up and photomultipliers was used to induce and measure NADH fluorescence on the cortical surface. The aim of the study was to assess the suitability of the laser induced spectroscopy for in vivo and on-line measurement of NADH in neuroscience and particularly for the assessment of neuronal metabolism. Changes in cerebral blood flow may affect fluorescence measurement. To assess the consequences of alterations in blood flow, the vasodilators glyceryl trinitrate and nimodipine and the vasoconstrictor endothelin-1 were applied. The induced hemodynamic changes were verified by colour Doppler sonography. The tests using the vasodilators showed that an increased blood flow in the brain increased not only NADH fluorescence but also the scattered light measured. The vasoconstrictor caused opposite effects. Insertion of a compensation method (subtraction of the scattered light) allowed the exclusion of hemodynamic artifacts. Effects of changes in the cellular metabolism were induced by sodium cyanide, an inhibitor of the mitochondrial respiratory chain, or by 2,4-dinitrophenol (2,4-DNP), an uncoupler of the oxidative phosphorylation. Sodium cyanide induced a transient increase of NADH fluorescence and 2,4-DNP decreased intracellular NADH fluorescence. Furthermore, the repercussions of cortical spreading depressions (CSD), a response of the brain to noxious stimuli, on cortical NADH fluorescence were determined. A single CSD decreased cortical NADH fluorescence for about 1 min, followed by a 5- to 10-min increase. The changes in NADH levels seem to correspond with the excitation and inhibition of neuronal metabolism, respectively. In summary, the measurement of NADH fluorescence using the laser technique allows the determination of changes in oxidative phosphorylation with high regional selectivity and time resolution.