The middle cerebral artery occlusion model is widely used to simulate ischemic stroke in mice. This model allows to investigate a temporal progression of ischemic damage, evaluate effectiveness of neuroprotective agents, and explore the role of a specific genes and pathways in stroke pathology with a final aim of finding new potential treatments. The implementation of neuroimaging techniques, such as MRI, allows to make better diagnosis, improve the assessment of outcomes, and reduce the number of animals needed in each study.
This protocol first a clear and detailed explanation of the most commonly used technique for inducing transient middle cerebral artery occlusion. Moreover, it implements the technique by introducing the recession modification of anesthesia with during occlusion time, reducing the detrimental effect of prolonged anesthesia time. To begin, position the anesthetized mouse on the operating table.
Maintain body temperature using a rectal probe connected to a heating pad. Make an incision on the skin of the head in the direction of the sagittal suture, starting from the ears to the area between the eyes. Retract the skin and remove the periosteum on the right side of the skull.
Then find the coordinates, 2.5 millimeters lateral from bregma. Apply cyanoacrylate to the doppler holder and attach it for laser doppler flowmetry. After the glue has dried, connect the doppler probe.
Then check for the correct signal readout for assessing the cerebral blood flow. To perform artery occlusion, turn the mouse over to the supine position and fix it to the surgical table with medical tape. Then, make a midline incision on the neck.
Pull the skin laterally, followed by pulling away the salivary glands using retractors. This exposes the carotid territory. Identify the vascular anatomy of the common carotid artery, or CCA, the ICA and the ECA as well as the different arteries derived from them.
For easier handling, detach CCA, followed by ECA from the connective tissue. Then, cauterize the superior thyroid artery and detach ICA from the connective tissue. Check again to make sure the main arteries are fully detached from the connective tissue.
Next, wrap 6-0 silk sutures around the ECA at the maxillary lingual bifurcation. Then suture around the CCA. Place a vascular clip interrupting blood circulation from the ICA.
Make a small incision in the ECA close to the area where the tight knot is located. Insert the monofilament until the thick coating has completely entered the arterial lumen. Tighten the second knot to hold the monofilament inside the artery and to prevent the pressure exerted by the blood from pushing it out.
Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA. Advance the monofilament via the ICA until the point where the MCA branches out. The occlusion is reflected in an abrupt blood flow drop in the LDF readout.
After 40 minutes, anesthetize the mouse again and place it back on the surgical table. Remove the stitches from the neck. After 45 minutes of occlusion, loosen the knot holding the monofilament in place.
Pull slowly and gently on the filament and check that tissue recanalization occurs. Then, pull out the filament and tighten the knot to prevent blood loss. Clean the blood and untie the CCA knot.
Ensure that there is no arterial wall damage. Remove the retractors and reposition the muscles, glands and skin. Suture the skin using a 6-0 suture and apply disinfectant.
Disconnect the doppler probe and detach the holder. Finally suture and disinfect the skin of the head. The outcome of the transient middle cerebral artery occlusion procedure was evaluated using in vivo MRI neuroimaging, Lesion evolution assessed at different time points after reperfusion showed that progression of the lesion volume takes approximately two days to complete.
Behavioral tests, such as grip strength test, showed a loss of strength in mice after 24 hours of transient middle cerebral artery occlusion Also, upon stimulation in the corner test, the mice showed a preference to turn to the right side, which was ipsilateral to the lesion indicating a successful middle cerebral artery occlusion on the right side.
