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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
- Middle cerebral artery occlusion (MCAO):
- Place and fix the anesthetized mouse (with laser doppler flowmetry (LDF) probe attached to its skull) in a supine position and maintain its body temperature using a heat lamp throughout the surgery. Disinfect the neck with 70% ethyl alcohol.
- Make a paramedian skin incision in the neck using a scalpel; blunt dissect the soft tissues with forceps to expose vessels. Add one drop of saline to the exposed tissues to keep them hydrated.
- Dissect the common carotid artery from the surrounding tissue and the vagus nerve using ophthalmic forceps. Be careful not to damage the vagus nerve. Disinfect the skin of the neck again with iodophor after the common carotid artery is exposed.
- Place a microvessel clamp at the distal end of the common carotid artery and then tie a dead knot with 6-0 silk sutures at the proximal end. Place the clamp as proximal as possible for subsequent operations. Ensure that the length of the vessel between the clamp and the dead knot is as long as possible for subsequent operations.
- Make a temporary suture proximal to the clamp by tying a loose knot. Ensure that there is enough space between two knots for monofilament insertion.
- Make a small longitudinal incision between the two knots with micro-ophthalmic scissors. Ensure the incision is as close to the dead knot as possible for subsequent operations. Be careful not to cut off the vessel.
- Insert a 12-mm tip-blunted monofilament through the incision to enter the artery lumen and advance it a few mm. Tighten the loose knot around the tip of the monofilament and then remove the clamp.
- Advance the filament into the internal carotid artery (10 mm to occlude the middle cerebral artery) with ophthalmic forceps until the LDF software shows a sharp decline (>80% reduction from baseline blood flow) in blood flow. Record the start time of occlusion.
- Occlude the middle cerebral artery for 1 h. Cut off the LDF probe and put the mice in a 30 °C incubator for the duration of occlusion.
- Reperfusion and Acidic Postconditioning (APC):
- Re-anesthetize the animals with isoflurane as described above, 55 min after the beginning time of occlusion. Place and fix mice in a supine position. Open the neck incision and re-expose the common carotid artery.
- Gently pull out the filament with ophthalmic forceps after the occlusion period to achieve reperfusion and turn the temporary suture into a permanent one by tightening the knot.
- For acidosis treatment, change the gas inhaled by the nose cone to the mix of gases containing 20% CO2, 20% O2, and 60% N2 for 5 min at 5, 50, or 100 min after reperfusion. Close the incision with an interrupted surgical suture.
- Place mice in a 30 °C heat cage until mice recover consciousness and then return the mice to clean, individual cages. Provide mice with a Petri dish of water and moistened food. Monitor the mice closely after the surgery for excessive pain and death.