윌리스의 마우스 원을 분리하는 프로토콜

The overall goal of this dissection procedure is to isolate the murine circle of Willis to facilitate the study of the large vessels of the mouse cerebrovasculature. This method can help answer key questions in the study of cerebrovascular gene expression in various diseases that effect the cerebral vessels such as Alzheimer's disease and cerebral ameloid angiopathy. The main advantage of this technique is that the large vessels can be quickly and easily isolated without altering the vessel structure.

Begin by using Iris scissors to make a four centimeter-long incision in the abdominal wall and peritoneum of a lethally anesthetized mouse just beneath the ribcage. Next, make a few millimeter long incision in the diaphragm, continuing the incision along the entire length of the ribcage to expose the pleural cavity. Lift the sternum and clamp the tip with a hemostat, resting the hemostat on the neck.

Then carefully trim the adipose tissue connecting the sternum to the heart. Now pass a 15-gauge profusion needle through the left ventricle into the apex of the heart, and use scissors to cut one of the liver lobes to create an outlet for the perfusate. Perfuse the animal with 25 to 50 milliliters of PBS at a pump rate of 2.5 milliliters per minute for about ten minutes.

The liver should blanch as the blood is replaced with PBS. When the fluid from the liver is completely clear, stop the perfusion. To isolate the brain, use Iris scissors to make a midline incision along the skin from the neck to the nose.

Trim away the skin with the Iris scissors to expose the skull and to remove any residual muscle and adipose tissue. Next, place the Iris scissors'tips into one side of the foramen magnum and carefully slide the tips along the inner surface of the skull to the external auditory meatus. Make a similar incision on the other side of the foramen magnum, followed by a midline cut along the inner surface of the interparietal bone to the start of the sagittal suture.

Plant the closed Iris scissors in the frontal bone between the eyes in the sagittal suture, and open the blades to split the skull in two. Using forceps, grasp the olfactory bulbs and use the Iris scissors to cut off the nerve connections on the ventral surface of the brain. Then immerse the brain in a 60 millimeter Petri dish of ice-cold PBS.

To isolate the circle of Willis, transfer the dish onto an ice pack under a dissecting microscope and turn the brain onto its dorsal surface to visualize the circle of Willis. Use the small forceps to grasp the anterior cerebral arteries at the base of the olfactory lobes, and exert pressure to dissociate the arteries from the vessel continuum. Cut the middle cerebral arteries in the same way.

Then use the sharp ends of the forceps to grasp the middle cerebral arteries and lift up the beginning of the posterior communicating arteries, disconnecting both sets of arteries from the brain. Next, lift the anterior arteries, pulling them gently over the optic chiasm in an anterior dorsal direction. Remove the superior and posterior cerebellar arteries in the same way, followed by the displacement of the basilar artery.

Finally, use the forceps to gently tranfer the entire circle of Willis into a 60-millimeter Petri dish containing ice-cold PBS, and fix the vessels in place with small pins, using two forceps to carefully remove any remaining brain tissue. The circle of Willis should come out in one piece, and should be slightly transparent due to the absence of residual blood in the vasculature. The clean circle of Willis should specifically express vascular smooth muscle cell markers, such as Smooth Muscle Actin, Smooth Muscle-Myosin Heavy Chain, and Smoothelin.

Neuronal markers, such as myelin oligodendrocyte glycoprotein, or microtubule-associated protein 2, by contrast, should be only barely detectable in the circle of Willis. Further, the expression of the endothelial marker, E-Selectin, in non-circle of Willis neural tissue, is very similar to that obtained for circle of Willis samples, possibly reflecting the existence of a a brain-capillary network. Once mastered, this technique can be completed in two hours if it is performed properly.

While attempting this procedure, it is important to remember that mouse cerebral vessels are very fine and break easily. Thus, the dissection should be carried out very carefully, without rushing, to ensure that the entire structure is harvested in one piece. After its development, this technique paves the way for researchers in the field of inflammatory neural disease to explore cerebral vascular disorder in mouse model of Alzheimer's and related diseases such as as cerebral amyloid angiopathy.

After watching this video, you should have a good understanding of how to isolate the circle of Willis from a mouse.