マウスにおけるレーザードップラー流量計の指針と虚血性脳卒中に対する腔内中大脳動脈閉塞（MCAO）モデル

In this video article, we present a murn model of ischemic stroke known as middle cerebral artery occlusion or MCAO. In the original method of MCA occlusion by coi, a blunted suture is advanced through the common carotid artery to occlude the middle cerebral artery. This method results in a permanent occlusion of the common carotid artery.

A methodological modification by longa allows access to the middle cerebral artery through the external carotid artery. This method preserves the function of the common carotid and its distal circulation Among experimental ischemic stroke Models. The intraluminal filament middle rebe artery occlusion model in rats and in mice is one of the most frequently used models since its first description in rats, several modifications of the initial model have been published currently long as modification is the most prominent variation.

This model is less invasive and easy to perform both permanent and transient ischemia in a controlled manner. Intraluminal filament middle cerebral artery occlusion model involves inserting a monofilament into the internal carotid artery and advancing it until it blocks blood flow to the middle cerebral artery. This model provides reproducible middle strip artery territory infarctions, and allows reperfusion after retraction of the suture.

The size of infarct appears to be affected by many specific factors in this technique, such as the suture diameter, the coating of the suture with silicone or polyol lysine, or the length of insertion of the filament itself. In this video, we'll show how to perform this model in a stepwise fashion, as well as how to avoid many of the potential pitfalls and drawbacks regarding the use of this method. A 7.0 monofilament nylon suture will be used for MCA occlusion.

The end of the nylon suture should be blunted by close proximity to a heat Source. The blunted end should be Straight and smooth and approximately one to two millimeters in length. After blunting the end, the suture should be cut to a total length of approximately 11 to 13 millimeters.

The blunted tip of the suture is dipped in silicone until three to four millimeters of the end is coated and allowed to dry. All steps in suture preparation should be conducted in sterile conditions. After weighing the mouse, anesthesia is induced with 4%isof fluorine in an induction chamber.

Mouse is placed in a prone position on a gauze pad lying on a thermal blanket. To maintain a core body temperature of 37 degrees, anesthesia is maintained during the procedure by a face mask. The shaved head is cleaned with alcohol, Betadine and again with alcohol.

A one centimeter incision is made in the skin to expose the calvarium one millimeter posterior and five millimeters lateral to the bgma. A flexible micro tip is affixed perpendicular to the surface of the right parietal skull to monitor blood flow. In the MCA territory by laser Doppler flow imagery.

Returning the animal to a supine position, a midline neck incision is made with scissors between the manubrium and the jaw. The paired submandibular glands are bluntly divided to expose the underlying structures, the di gastric muscle, the sterno colleto mastoid muscle, and the trachea form. A triangle retracting these structures and further dissection reveals the common carotid artery, the vagus nerve, and the internal jugular vein cran the common carotid artery bifurcates into the external carotid artery and the internal carotid artery.

The occipital artery can also be viewed originating from the bifurcation region. In COE's method, the proximal common carotid artery is carefully separated and isolated from the vagus nerve. Three loose collar sutures are prepared around the common carotid artery.

Special attention must be given so that the vagus nerve is not caught within A suture. The external carotid artery Is identified and isolated. It is then cauterized two to three millimeters distilled to the bifurcation.

The most proximal collar suture on the common carotid artery is tightened to permanently occlude the blood flow. The flow occlusion is verified by a drop from baseline cortical perfusion values of approximately 50%The free ends of the suture are trimmed. A vessel clip is placed just before the common carotid bifurcation to avoid retrograde blood flow.

Next, an arterio is performed between the vessel clip and the ligature with micro scissors. This allows the heat blunted suture to be introduced into the common carotid and advanced till it reaches the vessel clip. The two remaining loose collar sutures are gently tightened to avoid backflow leakage through the common carotid.

The vessel clip is withdrawn and the blunted suture is gently advanced into the internal carotid artery. Until mild resistance is felt on the Doppler readout, an approximate 80%drop from baseline results. The two collar sutures are securely tightened to avoid retrograde bleeding.

The mouse can now be left in a state of ischemia for an appropriate period of time. To simulate reperfusion, the occluding sutures withdrawn after an appropriate interval. A jump in the Doppler reading indicates that collateral blood flow has been restored.

The suture is completely withdrawn, and the two collar sutures are completely tightened and trimmed in long method. A different route can be used to occlude the middle cerebral artery. First, the external carotid must be identified, isolated and cauterized as distally as possible, leaving a sufficient length for suture insertion.

The external carotid artery is then Divided and its Branches are cauterized and cut as well. The stump of the external carotid artery is retracted. Coddly Two loose collar sutures are prepared around the proximal internal carotid artery just above the common carotid bifurcation.

The common carotid artery is temporarily closed with a vessel clip. This results in a drop in perfusion values of approximately 50%just distal to the bifurcation. The internal carotid artery is similarly clamped with a vessel clip and arter otomy is performed near the end of the retracted stump of the external carotid using micro scissors.

The heat blunted suture is introduced via the arterio and the external carotid and advanced till it reaches the vessel clip on the internal carotid. The two loose collar sutures around the proximal internal carotid are gently tightened to avoid reflux blood flow, though not enough to traumatize the arterial wall. Then the vessel clip is removed from the internal carotid artery, the tri palatine artery diverges from the internal carotid artery.

As the suture is gently, advanced care must be taken to avoid redirecting into it. A second drop on the Doppler readout is observed. When the blunted suture reaches the middle cerebral artery, the collar sutures must again be tightened to avoid retrograde bleeding.

The mouse can now be left in a state of ischemia for an appropriate period of time. Retracting the blunted suture restores blood flow as indicated by a rise on the Doppler monitor. After the sutures completely removed, the stump of the external carotid artery is cauterized.

To prevent post-surgical bleeding care must be taken to preserve the patency of the common carotid artery. The collar sutures at the proximal internal carotid artery are then loosened and cut away, and the clip on the common carotid artery is removed to restore blood flow. This is indicated with a second jump on the Doppler reading.

Once the mouse regains consciousness, neurological deficits can be evaluated at various time points according to a five point scale. A score of zero delineates no observable neurological deficit score of one corresponding to a mild stroke is evidenced by failure to extend a four PA on the opposite side of the stroke hemisphere. A score of two corresponding to a moderate stroke is evidenced by circling in a direction opposite to that of the stroke hemisphere.

A score of three corresponding to a severe stroke is evidenced by falling in a direction opposite to the stroke hemisphere. A mouse with A score of four will demonstrate no spontaneous motor activity To obtain a measurement Of stroke volume. The brain is removed from the skull at an appropriate time point.

After reperfusion, the brain is placed In a matrix slicer and the entire cerebral is sectioned coronal. At two millimeter interval, the Cerebellum and the olfactory bulbs are discarded. Each two millimeter section is immersed in 2%TTC solution for 20 minutes at 37 degrees in darkness.

Digital images of the brain sections show that the TTC stained living tissue red and left the infarcted tissue, a pale white for each section. The area of infarct labeled here as one. The area of the entire ipsilateral hemisphere labeled here as two, and the area of the contralateral hemisphere labeled here as three are all measured with the Image J program having measured the areas for all sections, irrespective volumes could be calculated by summing the area of values and multiplying by the slice thickness.

To find the infarct volume corrected for edema, the following formula is used. This volume is expressed as a percent of the ipsilateral hemispheric Volume. The MCO model provides Reproducible results.

However, a few procedural factors can interfere with the execution of the technique. The silicone coating on the end of the suture is important because it smooths the suture surface and lowers the risk of endothelial abrasion or a vessel perforation. Also, the suture coating may be modified to reflect different experimental goals.

Coating beyond three to four millimeter from the tip will occlude artery proximal to the middle cerebral artery increasing the total stroke volume. Color sutures should be applied with care so as not to restrict the vagus nerve and tie gently so the AAL Wall is not traumatized. The mouse body temperature can affect the extent of infarction, so this should be kept stable throughout the procedure.

We hope watching this video helps you navigate the surgical process and provides a solid starting point for your own experiments. Thank you for watching and good luck with your experiments.