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

The overall goal of this procedure is to directly visualize micro regional tissue hypoxia in the mouse cortex in vivo. This is accomplished by first preparing an open skull cranial window in an anesthetized mouse. Next fluorescent dye is injected intravenously to label the cerebral blood vessels.

Finally, concurrent to photon imaging of the intrinsic NADH fluorescence and the fluorescently labeled cortical microvasculature is performed. Ultimately, results can be obtained that show functional hypoxia in micro regions of brain parenchyma through visualization of the presence of the reduced form of nicotinamide, adenine dinucleotide. The main advantage of this technique over existing methods such as Clark style electrodes is that it, it gives a high special resolution over the large field of view, and it is also less invasive.

This method can be conducted on a standard applied two photo system with two fluorescent channels. Hi, I'm Steve Hurst, professor and chair of microbiology and immunology at the University of Rochester, demonstrating the procedure today will be Anita's son from the Kishka Laboratory, as well as Oxana Paulka, a research assistant professor from my laboratory, Dr.Kishka, who pioneered the NADH imaging technique, cannot be here today because he recently relocated to Germany. After preparing the surgical site with the necessary instruments that have been autoclave or disinfected with 70%ethanol and the mouse has been anesthetized with 5%isof fluorine, place it on a heating pad and apply a face mask to deliver 1.3 to 1.5%isof fluorine in air.

Check the level of anesthesia by performing a tail pinch. Next, insert a rectal probe and continuously measure the animal's body temperature throughout the procedure. Apply artificial tear gel to the mouse's eyes to prevent them from drying.

Then remove the hair from the head and both thighs apply hair remover to the thigh for two minutes to remove the remaining hair, the thigh will be used for oximetry. Finally, disinfect the scalp using a 10%povidone iodine solution and 70%ethanol. To make a cranial window starting five millimeters coddled to the skull with scissors.

Make an incision in the scalp and advance one centimeter. Move the skin to the sides to expose the skull To remove the membranes on top of the skull. Apply 10%ferric chloride solution.

Wipe away the excess solution and scrape the membranes away with a 45 degree angle. Number five, tweezers Using a dissection microscope, identify the area of interest. Apply a thin layer of rapid glue around the edges of the window of the head plate.

Position the head plate in such a way that the area of interest is exposed in the window and apply light pressure on the head plate. Apply a small amount of dental cement to polymerize the glue rapidly. Hold for 10 seconds while the glue is polymerizing.

Apply a small amount of rapid glue to the edges of the window to seal the head plate and the skull. Then screw the head plate to the animal holder under the dissection scope. Using the micro torque two drill set at 6, 000 RPM and the IRF 0 0 5 drill bit.

Remove all extra glue from the skull and from the head plate. Then set the drill at a thousand RPM and start drilling the skull by making a circle inside the head plate window with a diameter of about five millimeters. Use light sweeping movements as if you are drawing with a very soft pencil.

Stop drilling every 20 to 30 seconds and used compressed air to remove bone dust. As the drilling progresses, a burrow is formed around the intact skull in the center. Take care not to poke through the thin skull, but to make sure this burrow is of even thickness.

In addition, be extra careful around large blood vessels. They should not be compromised, and if possible, not even touched With one prong of a tweezers, lift off the bone in the center of the window and apply a drop of artificial cerebral spinal fluid on the window. Using the corner of a Kim wipe, gently wipe away the artificial cerebral spinal fluid using 45 degree angle.

Number five, tweezers. Locate a region of damaged dura along the edge of the window and lift it up to remove it. If bleeding occurs, apply a drop of artificial CSF and wait for two to three minutes for minor bleeding to stop.

Next, prepare 10 milliliters of 0.07%low melting aero dissolved in artificial cerebral spinal fluid. Bring the melted aeros to body temperature. Wipe away the excess artificial cerebral spinal fluid from the surface of the brain.

Pour the aeros in the window and cover it with a glass slide. Then press the glass gently to make contact between the glass and the head plate and wait for 10 to 20 seconds until the aero is solid. Using tweezers and soft tissue paper, remove excess aero from the glass cover.

Put a small amount of rapid glue around the glass to glue it to the head plate. Then apply a small amount of dental cement to solidify the glue to inject into the femoral vein. Turn the animal partially over to expose the inside of the left thigh.

Use tape to secure the animal. In this position, apply antiseptic scrub, and then a hundred percent ethanol to the skin. Next, make an incision along the medial thigh from the knee to the pubic synthesis.

If necessary, separate the soft tissues by blunt dissection. Fill a one milliliter syringe with 130 microliters of Texas red. Attach a new 30 gauge needle and bend it carefully at a 30 degree angle, keeping the bevel up.

Fill the needle with Texas Red Solution under a dissection microscope, insert the needle into the femoral vein and inject a hundred microliters of Texas red. Remove the needle and lightly press the vein with gauze to stop the bleeding. Close the skin using four oh suture for continuous monitoring of blood oxygen saturation.

Place the oximeter probe on the right thigh from which hair was previously removed. To begin imaging, transfer the surgically prepared mouse to the microscope stage. Take an initial picture of the cranial window site using Bright Field illumination at four x magnification to use as a reference map for registration with higher magnification to photon anies.

Zoom in on the area of interest using 10 x magnification. Select an area of interest in cortical layers. One or two start to record the time series.

We recommend using an average of three to five frames to increase the quality of the image. Next, induce hypoxemia by adding 50%nitrogen to the air that the animal is breathing. This will bring the oxygen level to 10%Verify hypoxemia by monitoring the oximeter data.

Continue to record the time series through hypoxemia for about 30 seconds and after normal oxygen level is restored. Collect data for calculation of the oxygen distribution by detecting, measuring and quantifying the area of hypoxia and oxygenated tissue cylinders around the blood vessels. To determine the croak tissue cylinder radius R, measure the distance between the center of the blood vessel and the boundary at which high NADH fluorescence is detected.

Refer to the written protocol for data processing methods. This figure shows concurrent NADH an geography images from video recorded during transient hypoxemia, the inspired oxygen concentration was lowered from 21%to 10%for a period of three minutes. This level of experimentally induced hypoxemia is sufficient to induce severe hypoxia in the cerebral cortex.

Hypoxia led to an increase in NADH fluorescence shown in green initially in the areas furthest from the arterial blood supply. Note, the sharp NADH tissue boundaries, which represent observable tissue boundaries of oxygen diffusion from the cortical microcirculation shown in red. Here is a concurrent image of the fluorescently labeled microvessels of the cortex in red, and the intrinsic NADH fluorescence from the gray matter of the cortex in green, the NADH fluorescence intensity distribution is binary.

There are areas with uniformly low NADH fluorescence and areas with uniformly high NADH fluorescence. The yellow lines represent the tissue boundaries between the areas with low and high NADH fluorescence. These tissue boundaries exhibit a spatial relationship to the nearby microvessels.

This relationship is determined by oxygen diffusion from the microvessels into the tissue. In some instances, the cylindrical nature or the CRO tissue cylinder of these tissue boundaries can be directly recognized as indicated by the blue lines. Here, the tissue boundary is of circular shape with a concentric central blood vessel Once mastered.

This technique can be done in 1.5 hours if it is performed properly. While attempting this procedure, it's important to remember to glue the cover glass to the metal plate to prevent vertical movement of the cortical surface due to edema. Following this procedure, additional analyses can be performed such as measurements of functional hyperemia with the goal of addressing additional questions such as the relationship between tissue hypoxia and cerebrovascular coupling.

After watching this video, you should have a good understanding of how to visualize micro regional tissue hypoxia in mouse cortex.