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

Summary

This protocol enables real-time monitoring of brain endothelial nicotinamide adenine dinucleotide metabolism in living mice using endothelial-targeted biosensor expression, cranial window preparation, and two-photon imaging.

Abstract

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.

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is a central cofactor for redox balance, mitochondrial activity, DNA repair, and stress-responsive signaling. In the cerebral vasculature, endothelial NAD⁺ homeostasis is increasingly recognized as an active regulator of barrier integrity, vascular tone, and neurovascular coupling rather than a passive metabolic readout¹. Disruption of endothelial NAD⁺ metabolism has been linked to aging-associated blood–brain barrier (BBB) leakage and impaired neurovascular homeostasis, highlighting the need for methods that can resolve endothelial metabolic dynamics directly in li....

Protocol

All procedures were approved by the Animal Ethics Committee of the University of Health and Rehabilitation Sciences under the project entitled “Mechanistic Study of Metabolic Probe-Based Observation of Blood–Brain Barrier Integrity” (approval number KFDX: NO. 2023-1023). Perform all surgeries under aseptic conditions and provide perioperative analgesia and supportive care according to the approved animal protocol. In the representative workflow, administer buprenorphine hydrochloride (0.05–0.1 mg/kg, subcutaneously) before or immediately after surgery and then every 8–12 h for 24–48 h as needed. Recover mice in a warme....

Results

A successful preparation yields a transparent and mechanically stable cranial window suitable for longitudinal imaging. Figure 1A summarizes the procedural workflow from anesthesia and skull exposure through craniotomy, coverslip placement, headplate fixation, and post-operative recovery. Figure 1B presents representative serial photographs of the major surgical steps. Windows suitable for imaging exhibit a clear optical path, visible cortical vasculature, an in.......

Discussion

This protocol provides an end-to-end workflow to monitor brain endothelial NAD⁺ metabolism in vivo. Key determinants of success include: (i) window clarity and stability, including minimization of thermal damage and control of bleeding during drilling; (ii) endothelial specificity and adequate sensor expression achieved through appropriate selection of the AAV capsid, promoter, dose, and expression interval; and (iii) consistent imaging and analysis settings, particularly when performing longitudinal .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,2,2-Tribromoethanol (Avertin)	Sigma-Aldrich	T48402	CAUTION: Toxic/harmful chemical. Used to prepare Avertin anesthesia solution for cranial-window surgery. Typical working solution: 1.25% (12.5 mg/mL), administered intraperitoneally at 200–250 mg/kg.
2-Methyl-2-butanol	Sigma-Aldrich	152463	CAUTION: Toxic/harmful chemical. Solvent used for preparation of concentrated Avertin stock solution. Protect from light and discard preparations showing precipitation or discoloration.
Absorbent swabs	Winner Medical	Sterile medical cotton swabs	Used for hemostasis, skull drying, irrigation, and surgical-field maintenance.
AAV-X1.1-Cdh5-cytoplasmic NAD+ biosensor-SV40 pA	Shanghai GeneChem Co., Ltd.; Addgene sources for plasmids	GeneChem construct GCPV5052075; Addgene #186787; Addgene #196836	Endothelial-targeted NAD+ biosensor packaged in AAV-X1.1. Expression cassette: Cdh5 promoter–cytoplasmic NAD+ biosensor–SV40 poly(A). Representative injection dose: 2 × 10¹² vg/mouse. Store aliquots at −80 °C and avoid repeated freeze–thaw cycles. Record lot-specific qPCR titer, batch number, plasmid map, sequence verification, and certificate of analysis.
Analgesic agent (buprenorphine hydrochloride)	MCE	HY-B0071	Perioperative and postoperative analgesia. Representative dose: 0.05–0.1 mg/kg subcutaneously every 8–12 h for 24–48 h.
Anesthesia delivery system	RWD Life Science	R500 with R580 vaporizer	Used for isoflurane delivery during imaging. Typical oxygen flow: 0.5–1.0 L/min.
Anesthetic agent (isoflurane)	RWD Life Science	R510-22 series	Used for imaging anesthesia. Typical settings: 3–4% induction and 1–2% maintenance.
Coverslip	Custom-made	No catalog number	Sterile No. 1.5 glass coverslip sized to completely cover a 3 × 3 mm cranial window and sit flush on the skull rim.
Dental cement/adhesive	Sun Medical	Super-Bond C&B; VZB/JAP8147	Used for cranial-window sealing and titanium headplate fixation. Representative preparation: 1 scoop powder, 1 drop catalyst, and 4 drops monomer. Harden approximately 5–10 min at room temperature.
Dental drill	RWD Life Science	78001 microdrill	High-speed drill used for skull thinning and craniotomy. Use approximately half of maximum speed with frequent saline cooling.
Disinfectant solution	Winner Medical	Povidone-iodine/iodophor and 70% ethanol	Used for scalp preparation before surgery. Apply alternating disinfectants for three cycles.
Fine forceps	JZ Brand	JD1050/JD1070	Used for periosteum removal, tissue manipulation, and bone-flap lifting.
Gavage needle	RWD Life Science	20G, 38 mm, ball tip	Used for oral NMN administration.
Headplate	Custom-made	No catalog number	Titanium headplate with approximately 10 mm central opening compatible with the imaging holder.
Heating pad/homeothermic controller	RWD Life Science	69020 with 69023 pad	Used to maintain body temperature at approximately 36.5–37.5 °C during surgery and imaging.
Image acquisition software	Nikon	NIS-Elements AR/AR Analysis v5.42.06	Used for microscope control and image acquisition. Representative settings include 512 × 512 or 1024 × 1024 pixels, 1–3 µm z-step, and 1 s time-lapse interval.
Image-analysis software	NIH	Fiji/ImageJ v1.8.0 or later	Used for motion correction, ROI selection, background subtraction, intensity extraction, and CSV export.
Intravenous injection supplies	BD	329461	Sterile 1 mL insulin syringe with 29G needle for AAV, TMR–dextran, and NMN injections.
NMN (nicotinamide mononucleotide)	Selleck Chemicals	S5259	NAD+ precursor used for drinking-water, oral-gavage, and intravenous administration experiments.
Ophthalmic ointment	Local certified pharmacy	Not applicable	Used to prevent corneal drying during anesthesia.
Phosphate-buffered saline (PBS)	Servicebio	G4202-100ML or G4202-500ML	Sterile 1× PBS used for viral dilution, tracer preparation, tissue rinsing, and reagent preparation.
Respiratory-monitoring system	RWD Life Science	Integrated monitoring module	Used to monitor respiration and anesthetic depth during surgery and imaging.
Saline (0.9% NaCl, sterile)	Servicebio	G4702-500ML	Used for skull cooling, irrigation, tissue moistening, and solution preparation.
Stereotaxic frame/head holder	Custom-made	No catalog number	Used for stable positioning during cranial-window surgery and imaging.
Surgical microscope	Olympus	SZ61	Used during cranial-window surgery for skull thinning, craniotomy, and coverslip placement.
TMR–dextran (70 kDa)	Thermo Fisher Scientific / Invitrogen	D1818	Intravascular fluorescent tracer used for vascular reference labeling. Typical concentration: 10–25 mg/mL. Inject at 20 mg/kg and allow 5–10 min circulation before imaging.
Tomato lectin (DyLight 488)	Vector Laboratories	DL-1174	Used for post hoc vascular/endothelial validation.
Two-photon microscope	Nikon	A1R MP+ multiphoton microscope	Dual-wavelength imaging system using 920 nm excitation for the sensor channel and 1040 nm excitation for the TMR–dextran reference channel. Record laser power, detector settings, filters, and acquisition metadata for each experiment.
Water-immersion objective	Nikon	CFI75 Apochromat 25XC W 1300	25× objective, NA 1.10, working distance 2.0 mm, used for in vivo two-photon imaging.