Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

The overall goal of this procedure is to make a permanent occlusion of the distal middle cerebral artery of a mouse with a resulting infarct that is mainly in the cortex and relatively about as large as human strokes. This is accomplished by first scoring baseline for limb use asymmetry before surgery. The next step of the procedure is to perform the stroke induction surgery by electrocoagulation of the distal middle cerebral artery.

After the surgery, focal sensory motor deficits are tested for by scoring four limb use asymmetry.Again. The final step is to analyze the infarct volume using crestal violet staining. Ultimately, the resulting cortical lesions are moderately sized with small variation, which makes this technique a valuable model for basic and translational stroke research.

The main advantages of this technique over other common stroke models are that a mortality rate in this model is minimal, the infarct volume and localization. The neocortex represent the majority of human strokes and it is highly reproducible. Generally, individual news to this method, we'll struggle first with the gentle removal of the skull to minimize mechanical lesions.

Secondly, the correct identification and proper correlation of the distal middle sal artery is critical to this model. First, inject analgesics 30 minutes before starting the operation. Turn on the heat blanket for the mouse and set it to 37 degrees Celsius for the operation.

Next, ensure that the anesthetic gas composed of 70%nitrogen, 30%oxygen, and iso fluorine is ready. Then expose the mouse to 4%ISO fluorine in an induction chamber until it stops moving. Then position the mouse on the heat pad and administer isof fluorine at 4%for one more minute.

After the minute, change the isof fluorine rate to 1.5%Now, ensure that the mouse is sufficiently anesthetized with a toe pinch, and if so, apply dafan ethanol to the eyes using scissors. Begin the surgery with a one centimeter incision between the ear and eye. Separate the skin and identify the temporal muscle.

Now on the high frequency generator, set the function to coagulation the mode to bipolar the power to 12 watts and attach the forceps. At the preparation, add a drop of saline to the exposed temporal muscle. Then use the electrocoagulation forceps to detach the temporal muscle from the skull at its apical and distal parts.

The result is a loosened muscle flap. Now identify the medial cerebral artery or MCA below the transparent skull. It is in the rostral part of the temporal area, dorsal to the retroorbital sinus.

Use a drill to thin the bone directly above this artery until it is fluent. Keep the skull moist during the drilling using saline. Now using very fine forceps, carefully remove the bone to expose the MCA branch.

Change the high frequency generator to seven watts. Now coagulate the artery with the electrocoagulation forceps by touching it gently without grasping. Coagulate the artery both proximally and distally to the bifurcation in the case of an anatomical variation without the bifurcation of the MCA, coagulate the artery as proximal as possible at two sites, about one millimeter apart after the coagulation.

Wait 30 seconds and then gently touch the artery with blunted forceps. Next, relocate the muscle to its position covering the bur hole. Suture the wound and place the animal in a nursing box at 32 degrees Celsius to recover from anesthesia and return it to the cage.

In general, it takes five to 10 minutes for the animal to recover from anesthesia. The cylinder test checks for favoring of one for limb over the other. Place the animal in a transparent acrylic glass cylinder flanked by two mirrors at a right angle.

Video the animal for five minutes opposite the vertex of the two mirrors on the other side of the cylinder. After each trial, wipe down the cylinder and tabletop with 60%ethanol. Later, perform a frame by frame analysis.

Score the assay by counting contact of one four paw to the cylinder while fully reared, and also by counting landing with one four limb. After a full rear score, at least 20 contacts of one four limb per trial cryo section, each brain serially into 20 micrometer thick sections taken at 400 micrometer intervals. Collect sections on slides to store at minus 20 degrees Celsius.

Using this sectioning protocol, all the sections covering the infarct area from one brain can be placed on one slide. Stain the sections using a standard crestal violet protocol to delineate the area of the infarcted tissue of each brain section. The slides can then be mounted, scanned, and analyzed using the Swanson method for edema.

Correction mortality due to the MCA occlusion surgery was less than 5%due mainly to subarachnoid, hemorrhaging or incorrect anesthesia. None died during the period. For this analysis for PA use, asymmetry was measured using the cylinder test after stroke induction, and compared to baseline values obtained before the MCA occlusion, significant limb asymmetry was detected at 24 hours, which partially recovered during the first week after the stroke, a week after the stroke.

Induction infarct voltri with crestal violet staining measured mean infarct volume at 15.4 cubic millimeters, thereby representing approximately 12%of one brain hemisphere. The standard deviation of this model is very low. In addition, the infarct location had minimal variability.

Once maade, this operation can be finished within 10 minutes if it's performed properly. Besides the demonstrated cylinder tests and the crystal violet staining, other established behavior tests and histological methods can be used to further characterize the outcome and address specific research questions. After watching this video, you should have a good understanding of how to perform a permanent distal MCA occlusion in mice.

We strongly recommend using standard operating procedures and detailed documentation to maintain the reproducibility of the method.