Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Abstract

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg 1 . We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 μm. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke. Video LinkThe video component of this article can be found at https://www.jove.com/video/3466/ Protocol Preparing the animal for imagingNote: We use adult c57BL mice. Different transgenic strains can be used depending on the research question. Microvascular surgeries and pulse oximetry are facilitated in mice with white fur (e.g. FVB strain).1. Prepare the surgical site at the bench. All surgical instruments need to be autoclaved or disinfected with 70% ethanol. 2. Insert a rectal probe, and continuously measure the animal's body temperature throughout the procedure 3. Anesthetize the mouse using initially 5% isoflurane. In 15-30 s, when the animal is not moving, place it on a heating pad (37 °C), and apply a face mask for the continuous delivery of air containing 1.3-1.5% isoflurane). Make sure that the animal is not responding to a pain stimulus (e.g. tail pinch). 4. Use artificial tear gel to cover the mouse's eyes, to prevent exposure to dry air. 5. Using an electric razor, remove hair from the head and from both thighs. Apply hair remover (e.g. Nair) to the thigh for 2 min, and then wipe it carefully to remove the remaining hair. This thigh will be used for oximetry. 6. Disinfect the scalp using a 10% povidone-iodine solution and 70% ethanol. Preparing the open skull cranial window1. Starting 5 mm caudal to the skull, make an incision in the scalp with scissors, and advance one centimeter. Move skin to the sides to expose the skull. 2. To remove the membranes on top of the skull apply 10% ferric chloride solution to the top of the skull. Wipe...

Monitoring NAD(P)H by an ultrasensitive fluorescent probe to reveal reductive stress induced by natural antioxidants in HepG2 cells under hypoxia

“…Previous studies have shown that hypoxia can enhance NAD(P)H levels. 36,37 Here, we investigated the imaging ability of TCF-MQ for NAD(P)H in HepG2 cells under hypoxia (1% O 2 ) and normoxia (20% O 2 ), respectively. HepG2 cells were cultured under hypoxic or normoxic conditions for 12 h, and then incubated with 10 mM TCF-MQ for 5-25 min.…”

Identification of potential mediators of oxidative damage and targets for novel treatments for chronic kidney disease

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Comparação dos efeitos da ressuscitação com Ringer lactato, solução salina hipertônica e terlipressina sobre a perfusão e oxigenação cerebral em modelo experimental de choque hemorrágico

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