Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice

We present a reproducible workflow for real-time visualization of nicotinamide adenine dinucleotide (NAD⁺) biosensor-channel responses in brain microvascular endothelial cells in living mice using cranial-window two-photon microscopy and an endothelial-targeted fluorescent NAD⁺ sensor. The protocol includes (1) adeno-associated virus (AAV)-mediated expression of the NAD⁺ sensor selectively in cerebrovascular endothelium using AAV-X1.1 under the Cdh5 promoter (vascular endothelial cadherin), (2) surgical preparation of a stable 3 mm × 3 mm cortical cranial window, and (3) dual-wavelength two-photon imaging to simultaneously capture the 920 nm-excited green sensor-channel signal and the 1040 nm-excited intravascular tetramethylrhodamine (TMR)-dextran reference channel. The TMR-dextran channel provides a vascular lumen reference and supports vessel selection, motion assessment, and vascular-integrity evaluation. As an application example, we describe nicotinamide mononucleotide-associated changes in endothelial sensor-channel fluorescence following drinking-water, oral-gavage, or intravenous delivery. This protocol emphasizes critical steps, recommended viral-dosing and titer considerations, troubleshooting, and quantitative-analysis strategies, enabling laboratories to implement in vivo monitoring of cerebrovascular NAD⁺ biosensor-channel dynamics for studies of neurometabolism in health and disease.