Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice.
R, Z., Y, L., X, M., H, C., M, Z., W, S., H, C., J, D., J, Z., & L, F. (2026). Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice.. Journal of visualized experiments : JoVE. https://doi.org/10.3791/71358
R Z, Y L, X M, H C, M Z, W S, et al. Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice.. Journal of visualized experiments : JoVE. 2026; doi: 10.3791/71358
R Z, Y L, X M, et al. Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice.[J]. Journal of visualized experiments : JoVE. 2026. DOI: 10.3791/71358.
@article{r2026,
author = {Zhan R and Li Y and Meng X and Cui H and Zhao M and Shan W and Cao H and Du J and Zhang J and Fang L},
title = {Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice.},
journal = {Journal of visualized experiments : JoVE},
year = {2026},
doi = {10.3791/71358},
note = {PMID: 42574365},
}
TY - JOUR AU - Zhan R AU - Li Y AU - Meng X AU - Cui H AU - Zhao M AU - Shan W AU - Cao H AU - Du J AU - Zhang J AU - Fang L TI - Real-Time Two-Photon Imaging of Brain Endothelial NAD+ Metabolism in Mice. T2 - Journal of visualized experiments : JoVE PY - 2026 DO - 10.3791/71358 AN - PMID:42574365 ER -
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.