Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

In the present research, we aim to provide a comprehensive and detailed protocol for establishing a medial cerebral artery occlusion model in C57BL/6NJ mice using transcranial electrode calculation and the evaluating the subsequent neurological behavior and histopathological features. The medial cerebral artery, or MCA, is recognized as the primary set of ischemic stroke in humans. The MCA occlusion methods, including craniotomy surround technique, focal chemical introduction, and for morphological occlusion, have been expensively employed to induce focal cerebral ischemia in rodents.

Compared to other ischemia stroke models, this model has the advantage of less surgical invasiveness, smaller infraction size, and higher survival rate, rendering it more suitable for investigating the long-term functional recovery after ischemic stroke. Overall, the current approach effectively generates a reliable experimental ischemic stroke model that exhibits high survivability and excellent repeatability. Consequently, this methodology serves as an invaluable tool for both foundation and translational research in the field of stroke.

In the future, we will employ behavior paradigms, such as novel object recognition and water maze, to investigate the dynamic alterations in long-term learning and memory capabilities among distal MCA-occluded mice. This will enable us to ascertain the viability of utilizing them as animal model for cognition impairment studies post-stroke. To begin, wipe the heating surgery board with 75%ethanol and set the temperature to 37 degrees Celsius.

Place the anesthetized mouse in a right lateral position on the board, with the head positioned away. To protect the cornea from drying, apply eye ointment. After shaving the fur from the left orbit to the left ear canal, use a depilatory cream to remove the remaining fur.

Disinfect the surgical area three times with povidone iodine and 75%ethanol. Perform a toe pinch assessment. After making a vertical incision between the left orbit and the left ear canal, retract the soft skin tissue to expose the temporal muscle.

Using straight micro forceps, separate the apical and dorsal segments of the temporal muscle from the skull. Identify the Y-shaped bifurcation of the middle cerebral artery, or MCA, beneath the temporal bone. Then, using an electric cranial drill, thin out the skull to make a bone window until the dura mater becomes visible.

Remove the dura mater above the MCA with curved micro forceps. Using electric coagulation forceps, coagulate the artery at the sites proximal and distal to the bifurcation. Monitor the blood flow of the left MCA cortical branch in a laser speckle blood flow meter.

Then, suture the muscle and the skin separately with polyglycolic acid sutures and apply diclofenac sodium gel and mupirocin ointment to the skin incision. Place the mouse in a recovery chamber. To perform the grip strength test in the previously developed distal MCAO model, grasp the posterior section of a mouse tail and gradually lower the mouse until it holds the horizontal bar with both forepaws.

Keeping the body horizontal, pull the mouse backward at a constant speed of two centimeters per second. When the mouse releases its forepaws from the bar, record the peak force in grams. For the pole test, place the mouse vertically on the top of a wooden pole.

Record the time taken by the mouse to turn around and the total time to climb down the pole with a 60-second cutoff time. To conduct the adhesive test, attach a patch of adhesive tape to the right forepaw of the mouse. Put the mouse back into the rearing cage and record the time taken by the mouse to remove the adhesive with a 60-second cutoff time.

To conduct a cylinder test, wipe an open top clear glass cylinder with 75%ethanol and place the mouse in it. Using a camera, record its spontaneous standing exploratory behavior for three minutes. The distal MCAO mice showed a significant reduction in grip strength and contralateral forepaw usage rate as compared to the sham-operated group.

Further, the occluded mice showed a significant increase in the descent time in the pull test and in the time taken to remove the adhesive. To begin, obtain the dissected brain of the euthanized distal MCAO mouse model after behavioral testing and place it in a minus 20 degrees Celsius freezer for 20 minutes. Then, position the brain into a one-millimeter brain matrix, and using microtome blades, slice the brain into two millimeter thick coronary sections.

Transfer the brain sections to a 24-well culture plate and add 2%TTC to cover the brain tissue. Incubate the plate at 37 degrees Celsius for 30 minutes. Then, carefully fix the brain section in 4%paraformaldehyde solution for 15 minutes.

Using a scanister, perform optical scanning of the brain sections. The histological analysis of the brain revealed the disordered arrangement of neuron cells and a significant reduction in the density of NeuN-positive cells in the peri-infarct area of the distal MCA-occluded mice.