The overall goal of this procedure is to produce a mouse model of focal cerebral ischemia by blocking the middle cerebral artery with an intraluminal thread. This is accomplished by making a midline neck incision and visualizing the common carotid artery. The next step is to tie ligatures around the common carotid artery and the external carotid artery clamps are used to keep them in place and to prevent bleeding.
The final step of the procedure is to introduce the filament into the circle of Willis. This blocks the branch of the middle cerebral artery, ultimately, neurological deficits. As a result of this, occlusion can be identified through observing changes in behavior.
One of the main advantages of proximal MCA occlusion via the filament technique is that it offers highly reproducible lesions in the stratum and in the cortex. Ischemia can be applied permanently and transiently, and furthermore, as it does not require craniectomy filamentous, MCA occlusion can be considered as more or less non-invasive. Generally individuals new to this method with struggling because of the delicate nature of microsurgical techniques Prior to the procedure, prepare the filament by cutting an 8.0 nylon filament into 11 millimeter lengths under the microscope.
The filament tip must be coated completely and evenly over a length of eight millimeters with a silicon mixture, the use of standard operating procedures is recommended for optimal results. The type of mouse used depends on the goal of the study. Generally, mice should be older than 10 weeks.
Use appropriate anesthetic agents in consult with veterinary staff. Body temperature of the mice is maintained at around 36.5 degrees Celsius during surgery with a heating plate, prepare the surgical site, disinfect the skin and the surrounding fur, and allow it to dry. Make a midline incision along the neck and gently pull the soft tissues apart.
The left common carotid artery is carefully dissected, free from the surrounding nerves without harming the vagus nerve, and a ligature is made using a 5.0 to 7.0 suture. Next, isolate the left external carotid artery and tie a second knot. After isolating the left internal carotid artery loosely tie a knot around it.
Using a 6.0 filament, the knot will be tightened later in the procedure. After obtaining a good view of the left internal carotid artery and the left tego palatine artery, both arteries, EC clipped to prepare for filament insertion. A small hole is cut in the common carotid artery before it bifurcates.
The monofilament prepared earlier is then introduced into the artery. The clipped arteries are opened while the filament is inserted into the internal carotid artery. To occlude the middle cerebral artery, the third knot on the internal carotid artery is closed.
To fix the filament in position, then apply lidocaine gel topically to the wound for pain relief. For sham operations, the filament is inserted and withdrawn immediately to allow instant reperfusion and immediately withdrawn to allow instant reperfusion administer 0.5 milliliters of saline subcutaneously as volume replenishment. To study the effects of reperfusion, open the third knot and remove the filament.
A set time period after ischemia, usually after 30 to 60 minutes. In this example, reperfusion is allowed 60 minutes after occlusion illusion, administer a second volume replenishing injection of saline. Finally shorten the remaining sutures and close the skin with a surgical suture.
Place the animals into a heated cage for two hours to recover after surgery, check the animals daily for signs of discomfort. Post-surgery, weight loss may occur, and mice should be provided with mashed food placed in a Petri dish on the floor. To encourage eating.
The food is replaced daily for seven days. The duration of blood flow restriction can lead to differences in motor and behavioral deficits. After 30 or 60 minutes of cerebral ischemia, most animals show decreased resistance.
S later push and circling due to a disturbance in locomotion, milder lesions manifest as a flexor position in the front limbs. These easily observable signs can be used as a basic score for the success of the operation. The resulting lesion can be assessed using either histology or magnetic resonance imaging.
As seen here, a 60 minute occlusion of the middle cerebral artery produces tissue pan necrosis in an area including both the striatum and the, whereas 30 minutes of ischemia causes mainly neuronal cell death, limited to the stri aum. Well-trained MCAO surgeons can master this technique in 10 minutes. While attempting This procedure, it's important to remember to follow standard operating procedures to ensure reproducible results and to control confounders.
