In this video, we will demonstrate a method of generating a cerebral infarct by endovascular occlusion of the middle cerebral artery in the rat. This is a widely used animal model in stroke research, which produces ischemia in the cerebral cortex as well as in subcortical structures, including the basal ganglia and the hypothalamus. This model can generate either permanent or transient cerebral ischemia.
Depending upon whether the intraluminal suture is left in place or removed after a defined period of time. Cerebral ischemia produces an infarct core in which cell death occurs, and a peri ischemic region term the penumbra in which some cellular recovery may be possible. Before we begin, let's take a moment to describe the steps we will follow.
Here we see a simplified diagram of the cranial circulatory system of the rat. In this surgery, we will be exposing the left carotid artery near the bifurcation of the common carotid artery or CCA into the external carotid artery, the ECA and the internal carotid artery. The ICA two small branches of the ECA, the occipital artery and the superior thyroid artery will be ligated using bipolar coagulation and the ECA will be tied off distally to these two branches Using a silk suture, microvascular clips will be placed onto the common carotid and internal carotid arteries.
A nylon monofilament suture with a silicone coated tip will then be inserted through an incision in the ECA and routed up into the ICA. This intraluminal suture is lightly tied in place with a silk suture around the ECA prior to releasing the micro clip from the ICA. The intraluminal nylon suture is then advanced approximately two centimeters until the silicone tip is occluding the origin of the middle cerebral artery or MCA.
The second micro clip is removed from the CCA and the period of occlusion is timed. Now that we have an overview of the procedure, let's move on to the surgery. Prepare the red as you would for any surgical procedure according to the animal care guidelines of your institution, we place the red under anesthesia using 5%isof fluorine for induction at a flow rate of one liter per minute oxygen and one liter per minute nitrous oxide, we reduce the isof fluorine concentration to one to 2%for maintenance.
Once an anesthetic plane is achieved, we apply artificial tears ointment to both eyes and shave the neck with clippers. The region is then disinfected with betine solution followed by 70%ethanol. This process should be repeated for a total of three cycles of Betadine and 70%ethanol rinses.
The red is placed in a sterile stocking and laid upon a thermal regulatory blanket. To prevent hypothermia. It is helpful to raise the neck slightly by sliding a 15 mil conical tube underneath the red's head.
This will aid in exposing the carotid arteries later. Finally, 0.2 mils of a 0.5%vacating solution is injected subcutaneously along the presumptive incision site in order to provide postoperative pain relief under an operating microscope. Make a midline incision using a number 10 scalpel blade.
The remaining dissection will occur to the animal's left side of the midline. Begin dissecting through the superficial fascia. This is done with a combination of both blunt and sharp dissection.
Extensive glandular tissue is visible laterally while the large sternal od muscle resides midline overline. The trachea use cotton tip applicators to dissect the gland laterally and the stern hi muscle medially. Underneath the glandular tissue, you will see the digastric and sternal mastoid muscles, which along with the midline stern hi muscle or muscle subtle triangle whose peak is circled.
It is within this triangle that the dissection will continue to the carotid artery. Use micro scissors to cut through the fascia covering the muscles. This will offer retraction of the muscles to expose the underlying vessels.
Cotton tip applicators can be used to retract the muscles and bring the common crowded, external, crowded, and internal crowded arteries into view. Continue working to expose the ECA in its branches. This will require careful sharp dissection of the vessels.
The goal of these steps is to free the ECA and ICA from underlying connective tissue. Continue to dissect the ECA ally. Note that the HI hide bone may be encountered and this will limit the extent of rostral dissection.
The vagus nerve, which appears white, is seen coursing along the lateral aspect of both common and internal carotid arteries, and we sharply dissected off the CCA and the ICA. Two branches coming off of the ECA are also sharply dissected. The first branch heading laterally is the occipital artery and the second branch heading medially is the superior thyroid artery.
Both the first and second branches off of the ECA are cauterized and subsequently cut. Cutting these branches allows for greater ease in mobilizing the vessels. Later in the procedure using a silk suture securely tie off the ECA as far distally as possible.
Place another silk suture loosely around the ECA proximal to the bifurcation of the common carotid artery. Make sure not to occlude the vessel with this tie as the endovascular suture used for middle cerebral artery occlusion. We'll be going through this portion of the ECA place microvascular clips on both the common carotid and internal carotid arteries.
That position somewhat removed from the origin of the ECA. Next, create a partial arter otomy between the two silk suture ties of the ECA. A small amount of residual blood may be released, but extensive bleeding likely indicates improper clamping with the microvascular clips.
In this case, be sure that no underlying tissue has been caught in the clip blades preventing full closure. The next step is to insert a three centimeter length of monofilament nylon suture into the ECA. It's helpful to mark the suture at two centimeters from its silicone coated tip.
These sutures are commercially available and the tip diameter and length of silicone coating can be custom designed to your specifications depending upon the age and weight of the rats that you will be using. For 300 gram rats, we use a 0.39 millimeter tip diameter and two to three millimeters silicone coating length on a four oh nylon suture. Introduce the occluding nylon suture into the ECA lumen down toward the common carotid artery.
Tighten the silk suture around the ECA stump to secure the intraluminal nylon suture and prevent bleeding. Cut the remaining portion of the ECA to free the stump and position the ECA stump below the bifurcation point such that it is roughly in line with the ICA. This will more easily allow the intraluminal suture to slide into the internal carotid artery.
Remove the microvascular clip from the ICA. Remember to open the clips slowly to check for bleeding before removing it completely. Continue advancing the nylon suture within the ICA lumen until it reaches the middle cerebral artery.
This length is typically around two centimeters and is the reason for placing a mark on the suture prior to insertion. After a variable length of nylon suture is inserted, resistance may be felt if resistance occurs with much of the nylon suture still present outside of the vessel, it likely indicates that the suture is entering the tego palatine artery. In this case, pull back and curve the suture slightly to continue along the ICA, which will run more immediately.
Note the change in position of the suture inside the ICA lumen in these two comparison shots. Proceed advancing the suture through the ICA until most of the sutures inserted and resistance is again felt at this point. The intraluminal suture has blocked the origin of the MCA.
Start the timer and record the occlusion. Start time. Remove the other microvascular clip from the CCA, close the incision quickly and carefully.
Place the red in a recovery cage. Check that the cage floor around the nose and mouth are free of bedding materials shortly before the occlusion period should end anesthetize the rat and reopen the incision by removing the closing sutures. Place a micro clip on the CCA as before.
Withdraw the occluding suture partway from the ICA until the suture end is visible through the ICA. Do not yet fully remove the suture. However, place another micro clip on the ICA above the location of the intraluminal suture completely remove the occluding suture and tie off the ECA stump.
Record the reperfusion start time. Remove the micro clips from the ICA and from the CCA moist in the region with several drops of sterile saline and close the incision with suture. We inject buprenorphine at a dose of 0.05 milligrams per kilogram body weight for additional postoperative analgesia.
We also administer five mils of saline intraperitoneal to provide hydration during the recovery stage. After the rat has recovered from anesthesia test for behavioral indication of infarction by holding the rat by the tail and observing whether the rat can turn to both sides in this elevated body swing test curling to one side only as expected. Rats exhibiting stroke symptoms may also circle to one side during open field movements.
Here we show results of 60 minutes of MCA occlusion in spontaneously hypertensive rats. Magnetic resonance imaging was performed 24 hours after MCAO and T two weighted images are shown. This rat developed a large infarct involving both cortical and steroidal regions.
The hyperintense area on the MRI is highlighted in yellow, although we don't show the results here. Infarcted areas visualized by MRI can be further analyzed by diffusion weighted imaging and apparent diffusion coefficient maps. The infarcted regions are also confirmed by histological staining of freshly isolated brain sections with trien tetra allium chloride or TTC dehydrogenase in living tissue convert the substrate to an insoluble red dye.
Thus the infarcted regions remain unstained and appear white. Note that infarct size can be affected by several factors including rat strain and animal size. We use spontaneously hypertensive rats as they produce more consistent infarct sizes than other commonly used strains such as frogg, dolly or wistar rats.
It is also important to standardize on a target rat weight to reduce variations in vessel diameter and thus in the resulting efficacy of MCAA occlusion. We hope that you have found this video helpful in performing the MCA occlusion technique in your own lab. Thanks for watching and good luck with your experiments.
