The overall goal of the Longa method is to reproduce ischemia at the root of the middle cerebral artery, or MCA, since ischemic strokes in humans most commonly occur due to the occlusion of the MCA. This method can help us answer key questions in the stroke field such as how to find a suitable therapeutic target for the treatment of this disease. The main advantage of the Longa method as opposed to alternative techniques is the survival rate.
There's also an increase in leukocyte-endothelial cell interactions and infarct volume with this technique. Demonstrating this procedure will be Shantel Vital, the manager for my laboratory. After inducing anesthesia in a male C57 black six mouse according to the text protocol, monitor the depth of anesthesia by a foot pinch and apply a sterile ocular ointment to prevent dryness.
Then, shave the animal's neck. Place the mouse in a supine position on a temperature-regulated mat and insert a rectal probe to monitor and maintain a body temperature of 36.5 plus or minus 0.1 degrees Celsius. Then, use 70%ethyl alcohol to disinfect the skin.
Next using Iris straight scissors, make a midline incision and retract the soft tissues to expose the vessels. Using Dumont forceps, dissect the common carotid artery, or CCA, and the external carotid artery, or ECA, from the surrounding tissue without damaging the vagus nerve. With 7-0 silk, make a temporary suture by tying a loose knot around the CCA.
Then, make a permanent suture around the ECA and the smaller vessels extending from it by tightly ligating the vessels. Now, make a suture around the ECA proximal to the CCA bifurcation. Then, place a microvessel clip around the internal carotid artery, or ICA, and the pterygopalatine artery, or PPA.
Using microdissecting spring scissors, make a small incision in the ECA as close to the permanent suture as possible and insert a 180-micrometer silicone-tipped monofilament. With the filament inserted, tighten the temporary suture around the ECA and remove the microvessel clip. Use the microdissecting spring scissors to cut the ECA between the permanent distal suture and the entry point of the filament.
Guide the filament through the ICA until resistance is felt in the MCA. Refer to additional details in the text protocol. After a 30-minute occlusion period, remove the filament by using Dumont forceps to gently pull it back and secure the suture around the open end of the ECA.
Remove the temporary suture around the CCA by carefully loosening the ligature to allow blood flow to resume through the CCA. Remove the retractors, and then use a continuous surgical suture to close the incision. Inject the mouse with one milliliter of saline subcutaneously for volume replenishment and with the analgesic carprofen for relief of pain and discomfort from the surgical procedure.
Observe the mouse throughout the recovery from the anesthesia in a dedicated cage heated to 30 degrees Celsius and place mashed chow in a Petri dish on the floor of the cage to encourage eating. Using an 18-point scoring system, neurologically evaluate mice after the appropriate reperfusion period assessing the general, motor, sensory and proprioception scores. A higher neurological score corresponds to decreased neurological function.
To measure bran infarct volume, after inducing anesthesia and monitoring the depth according to the text protocol, place the mouse in a supine position and use Iris straight scissors to cut the skin from the abdomen to the neck before cutting through the peritoneum. Using Dumont forceps, lift up the sternum and cut the ribs. Then, use two pairs of hemostats to open the left and right sides of the chest exposing the heart.
Insert a 26-gauge needle attached to a five-millimeter syringe into the left ventricle and cut the right atrium. Then with room temperature normal saline, or PBS, perfuse the heart until the fluid becomes clear. With the Iris straight scissors and forceps, remove the animal's head and carefully remove the brain from the skull.
Then, use a razor blade to cut off the olfactory bulb and the cerebellum so that the brain will fit into the matrix. After chilling the matrix, place the brain inside and set it on ice. Then with a razor blade, make a first cut in the brain two millimeters from the top and leave the blade in place.
With a second razor blade, make another two-millimeter cut behind the first. Then, remove the first razor blade with the tissue attached. Repeat this process until all the tissue has been sliced.
Place each segment in the order in which they were cut into one well of a 24-well plate containing 2%two, three, five-triphenyltetrazolium chloride, or TTC, and place the plate in a shallow water bath at 37 degrees Celsius for 20 minutes. After incubating for 10 minutes, turn over the slices. Then, place a small amount of 10%formalin into the wells of a new 24-well plate and transfer the segments into the wells of formalin in the order in which they were cut.
After scanning the segments into the computer, use ImageJ analysis software to analyze the infarct size as a percentage of the whole brain slice by outlining the infarcted area to generate an area measurement. Next, measure the entire contralateral hemisphere to determine the area. Finally, divide the infarct area by the area of the contralateral hemisphere and multiply it by 100 to determine the infarct volume.
As shown here, laser doppler flowmetry was used to confirm blood flow perfusion in the MCA territory before and after MCA reperfusion. When the temporary CCA tie is released after 30 minutes of ischemia for filament removal and reperfusion to occur, there is a surge in perfusion which reached just under 100%of baseline perfusion five minutes into reperfusion. Listed in this table are the neurological scores pertaining to the assessment of general, sensory, motor and proprioceptive deficits.
This graph illustrates that the score for 27 mice at 24 hours was 12.56 plus or minus 0.7 and remained high one week later at 11.50 plus or minus 1.5. A higher neurological score represents decreased neurological function. Infarct volumes showed similar patterns to the neurological score at 24 hours.
As presented here, 24 hours after MCAo, mice had large infarct volumes which were heightened one week post stroke. Once mastered, this technique can be performed within 15 minutes if it's done properly. While attempting this procedure, it's important to maintain the temperature at 36.5 degrees Celsius.
Following this procedure, other methods can also be performed such as cecal ligation and puncture. This enables us to answer additional questions such as the effects of sepsis before and after stroke. After watching this video, you should have a good understanding of how to induce ischemia by inserting a monofilament into the middle cerebral artery and removing it to allow for reperfusion.
Don't forget that working with instrumentation such as Iris scissors and forceps can be dangerous and precautions should always be taken.
