Inducing White Matter Stroke in a Murine Model Using a Nitric Oxide Inhibitor

Begin with an anesthetized mouse immobilized in a stereotactic frame with an exposed skull.

Mark the bregma, then drill a hole, beginning posteriorly at the bregma and extending anteriorly, just left of the midline.

Remove tissue layers to expose the myelinated axon-rich cerebral cortex, and apply saline drops to prevent tissue drying.

Attach a pipette filled with a nitric oxide inhibitor to an injector arm, which is positioned at an angle for precise injection.

Adjust the stereotactic coordinates and inject the inhibitor into the white matter of the cerebral cortex, holding the pipette briefly to prevent backflow.

Retract and repeat the injection at other coordinates.

Later, seal the hole and close the incision with adhesive.

The inhibitors bind to nitric oxide-producing enzymes in blood vessel endothelial cells, inhibiting nitric oxide production and constricting blood vessels.

This constriction inhibits blood flow, leading to neuronal damage and lesion formation, developing a white matter stroke.

Begin, by first affixing a pulled glass pipette to tubing attached to a vacuum line. Insert the pulled end into the L-Nio solution or L-Nio plus fluorescent tracer. Start the vacuum, and apply suction until at least 2 millimeter of the 0.5 millimeter diameter portion of the pipette is filled.

Put the filled pipette to one side. Then, place the mouse into a stereotactic apparatus equipped with a stereotactic microscope. After anesthetizing and prepping the mouse for surgery according to approved procedures, check the depth of anesthesia with a toe pinch. There should be no response.

Next, adjust the injection arm to 36 degrees. Affix a pulled glass pipette holder to the distal end of a low volume pressure injection system and attach it to the injection arm. After making a 1.5 centimeter midline scalp incision to expose the skull, dry the surface skull with a cotton swab.

Then, while looking through a stereotactic microscope at 1 to 3X magnification, use a micro point tool to remove any overlying periosteal tissue. Mark bregma with a fine point marker, then use a surgical drill equipped with a fine tip surgical drill bit to drill a 2 millimeter elliptical craniotomy, beginning posteriorly at bregma and extending anteriorly just left of the midline.

Remove bone fragments and overlying soft tissue so that the cerebral cortex can be visualized. Keep the cortical surface moist by intermittently applying drops of sterile saline. Next, affix the filled pipette to the injector arm. Align the distal end of the pipette with bregma, and zero the stereotactic coordinates.

Advance the pipette to the first injection point using the anterior, posterior, and medial lateral coordinates listed in table 1. Then, advance the pipette to the cortical surface and zero the dorsal ventral measurement. Slowly lower the pipette to the dorsal ventral coordinate.

Ensure that the stereotaxic microscope magnification is set to 3X and that the calibrated reticule can be clearly viewed. View the angled pipette from the side so that the air fluid meniscus has a sagittal view. The meniscus should appear in the same focal plane of both the inner and outer wall of the pipette.

Now, using a low volume pressure injection system, set at 20 PSI for 20 millisecond pulses, inject 100 nanoliters of L-Nio into the brain. After injecting the total volume, wait 5 minutes to prevent reflux up the pipette track. Then, withdraw the pipette and repeat the injection procedure at the second and third set of coordinates provided in table 1.

After the final injection, remove the pipette and place enough bone wax to fill the craniotomy site. Finally, approximate the edges of the scalp wound and bind with dermal adhesive.