Medicine
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Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability
Chapters
Summary February 23rd, 2016
Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.
Transcript
The overall goal of this procedure is to perform a permanent middle cerebral artery, or MCA occlusion in an elderly rat model. This is accomplished by first exposing and loosely looping the carotid arteries for later ligation. In the second step, the skull is drilled to reveal the MCA.
The vessel is then coagulated with forceps to initiate the occlusion. In the final step, the right carotid artery is permanently tied off and the left carotid is transiently occluded. Ultimately, T2 weighted magnetic resonance imaging is used to observe the size of the lesion and to confirm the success of the procedure.
This model allows for reproducible-sized infarcts in the middle cerebral artery area and sustained behavioral deficits in elderly rats. Before beginning the procedure, confirm the proper level of sedation by a lack of response to toe pinch in a 16 to 18-month-old female elderly Lister Hooded rat, and administer the appropriate analgesic. Next, shave the fur on the ventral neck and right hemisphere temporal regions of the animal, and use ethanol swabs to disinfect the exposed skin.
Place the rat in the supine position on a cork board covered with a sterile drape on top of a heating pad, and apply lidocaine cream to the shaved regions of the head and neck. Insert a rectal probe to maintain the body temperature between 36.5 and 37.5 degrees Celsius, and put ointment on the animal's eyes. Then inject the rat with 0.05 milliliters of atropine sulfate to reduce the tracheal secretions, and place the animal under a stereo microscope.
Begin the surgical procedure with a two centimeter central midline incision into the exposed neck, and move the salivary glands gently lateral to the trachea on both sides of the animal. Next, on the right side of the neck, loop a non-absorbable silk 5-0 suture through the skin overlying the exposed region, and use the sutures to gently pull the skin away from the incision. Using surgical tape, attach the sutures to the cork board to reveal the common carotid artery.
Then, using forceps, carefully blunt dissect the artery from the surrounding fascia. Once the common carotid artery is exposed, use fine forceps to reverse dissect the artery from the vagus nerve, taking care not to make contact with the nerve. Loop the vessel with a non-absorbable suture, and tape the ends of the suture together with surgical tape.
Then remove the sutures holding back the overlying skin, ensuring no interference with the suture looped around the carotid artery. Repeat the procedure on the left side as just demonstrated. After both arteries have been looped and the extra sutures have been removed, place a sterile, saline-soaked gauze into the wound to keep the tissues moist, and loosely suture the skin.
Place another saline-soaked gauze over the area to prevent further dehydration, and change the rat to a lateral position. Incise the skin at the midpoint between the right orbit and the external auditory canal. Next, blunt dissect the temporalis muscle to reveal the skull.
Then, using up to five elastic three millimeter hook retractors, retract and pin the skin to the cork board. Then place a thumb wheel adjusted, gravity-driven saline drip above the open site at the highest point of the skull near the ear, and set up an aspirator system with the nozzle at the lowest point to remove any bone debris and to clear any minor bleeding from the exposed site. Adjust the wheel to visualize the source of bleeding as necessary.
Now use a dental drill with a coarse 1.6 millimeter diamond-coated drill burr at approximately 8, 000 rpm to perform a five millimeter by five millimeter craniotomy on the exposed region, taking care to apply circular and lateral, but not downward pressure during the drilling. When it becomes completely transparent, use forceps to remove the bone. Then, using a homemade dural hook, carefully open the dura, taking care to avoid rupturing the large surface blood vessels.
Next, use a pair of angled jeweler diathermy forceps with 0.25 millimeter pointed tips to coagulate the MCA from where the inferior cerebral vein crosses to the point of the artery bifurcation, and then along the caudal branch of the MCA until the vessels are fully occluded. Then, to confirm the occlusion is complete, use microvascular scissors to cut the section of the occluded MCA that lies beneath where the inferior cerebral vein crosses the MCA. Now cover the exposed area with a saline-soaked gauze pad, and return the rat to the supine position.
Reopen the loosely tied suture on the neck to re-expose the carotid arteries, and tie a knot in the suture around the artery on the same side as the occluded MCA to permanently ligate the vessel. Then use a 13 millimeter stainless steel artery clip with approximately 125 grams of pressure to transiently ligate the left carotid artery for one hour. During the temporary occlusion, loosely suture up the incision in the neck, and place another sterile, saline-soaked gauze over the incision.
Next, suture the incision in the temporal region, followed by four five milliliter subcutaneous injections of saline. At the end of the temporary occlusion period, remove the clip and apply saline topically to the surrounding muscle. Then, using subcutaneous continuous stitching of absorbable 4-0 sutures, close the incision in the neck, and allow the animal to recover from the anesthesia for four hours in a 31 degree Celsius incubator.
If a rat exhibits weight loss after the surgery, inspect the top and bottom teeth. Then, with the animal in the supine position, place a one millimeter syringe barrel behind the teeth to protect the soft tissues, and use a handheld circular saw at a high rotary speed and slow, firm hand movements to trim the animal's teeth. To measure the infarct volume, 24 hours after the induction of the middle cerebral artery occlusion use structural MRI to obtain T2 weighted scans.
Then measure the cross-sectional area of the infarct in 20 volumes to obtain lesion volumes in a medical image display package, and multiply the sum of these areas by the thickness to obtain the total volume of the infarct. For percentage lesion volume calculations, acquire the volumes of the ipsilesional and contralesional hemispheres as well. To calculate the sample size for future studies, use power analysis software to determine the sample size calculations for estimating group sizes required to identify the treatment effects of different magnitudes.
In this experiment, the stroke outcome was assessed 24 hours and eight weeks after MCA occlusion as just demonstrated, with the infarct volume identified as the areas of the rat brain exhibiting a hyper-intense signal. In this representative experiment, at 24 hours the raw, unadjusted lesion volume was 62.8 millimeters cubed, with the lesion occupying 9.8 percent of the affected hemisphere. When corrected for brain swelling using Gerriets formula, this value was reduced to 4.5 percent.
Stroke severity was also measured using the Montoya staircase test, with an approximately 40 percent reduction in sugar pellet retrieval observed in the MCA occlusion stroke animals. When attempting this procedure, it's important to remember to carefully dissect the vagus nerves when manipulating the carotid arteries for ligation, as damage to these nerves can negatively affect the animal's recovery. Following this procedure, treatments can be given in order to answer additional questions, like how neuroprotection can affect the size of the infarct, and what treatments may be able to improve outcome after stroke.
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