Quantification of cytochrome c oxidase and tissue oxygenation using CW-NIRS in a mouse cerebral cortex

We provide a protocol for measuring the absolute concentration of the oxidized and reduced state of cytochrome c oxidase (CCO) in the cerebral cortex of mice, using broadband continuous-wave NIRS. The algorithm (NIR-AQUA) allows for absolute quantification of CCO and deoxyhemoglobin. Combined with an anoxia pulse, this also allows for quantification of total hemoglobin, and tissue oxygen saturation. CCO in the cortex was 4.9 ± 0.1 μM (mean ± SD, n=6). In normoxia, 84% of CCO was oxidized. We include hypoxia and cyanide validation studies to show CCO can be quantified independently to hemoglobin. This can be applied to study oxidative metabolism in the many rodent models of neurological disease.