In vivo measurement of NADH fluorescence lifetime in skeletal muscle via fiber-coupled time-correlated single photon counting

Nicotinamide adenine dinucleotide (NADH) is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS). NADH is autofluorescent, and its fluorescence lifetime can be used to infer metabolic dynamics in living cells. Fiber-coupled time-correlated single photon counting (TCSPC) equipped with an implantable needle probe can be used to measure NADH lifetime in vivo, enabling investigation of changing metabolic demand during muscle contraction or tissue regeneration. This study illustrates a proof of concept for point-based, minimally-invasive NADH fluorescence lifetime measurement in vivo. Volumetric muscle loss (VML) injuries were created in the left tibialis anterior (TA) muscle of male Sprague Dawley rats. NADH lifetime measurements were collected before, during, and after a 30s tetanic contraction in the injured and uninjured TA muscles, which was subsequently fit to a biexponential decay model to yield a metric of NADH utilization (cytoplasmic vs protein-bound NADH, the A(1 tau 1)/A(2 tau 2) ratio). On average, this ratio was higher during and after contraction in uninjured muscle compared to muscle at rest, suggesting higher levels of free NADH in contracting and recovering muscle, indicating increased rates of glycolysis. In injured muscle, this ratio was higher than uninjured muscle overall but decreased over time, which is consistent with current knowledge of inflammatory response to injury, suggesting tissue regeneration has occurred. These data suggest that fiber-coupled TCSPC has the potential to measure changes in NADH binding in vivo in a minimally invasive manner that requires further investigation.