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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.
1. Mouse Model of Middle Cerebral Artery Occlusion (MCAO)
- Use male C57BL/6 mice aged 6 weeks and weighing 22-25 g. Provide all animals with free access to standard chow and water, and house them in an environment with controlled temperature (22 ±1°C) and a 12 h light/dark cycle.
- Divide the mice into groups of six mice each, which should consist of sham-operated normal, control, and Glycyrrhizae Radix et Rhizoma (GRex) treatment groups.
- Perform MCAO surgery on the control and GRex treatment groups using a stereo-microscope.
- Induce inhalation anesthesia in the mice using 2% isoflurane in 70% nitrous oxide (N₂O) and 30% oxygen (O₂). Anesthesia is considered sufficient when the mouse becomes unresponsive to a mechanical stimulus applied to its tail. Maintain the body temperature of the mice at 36.5 ± 0.5 °C using a body temperature-holding blanket connected to a thermometer.
- Remove all the hair on the chests and necks of the mice by shaving, followed by the use of hair removal cream. Disinfect the surgical site of skin with betadine scrub alternating with alcohol twice, and then make an incision of approximately 2 cm long with a surgical blade in the center of the neck. Carefully isolate the left common carotid artery (LCCA), external carotid artery, and the branch of the internal carotid artery from surrounding connective tissues.
- Ligate the external carotid artery and the common carotid artery with a surgical suture (4-0 silk suture, half hitch knot) to temporarily block the blood flow into the internal carotid artery during the operation.
- Insert a silicon-coated nylon suture (8-0 monofilament, 11 mm long) through the internal carotid artery to the origin of the left MCA. Adjust the thickness of the silicon-coated part of the filament to a range of 0.10-0.12 mm.
- Measure the decrease in relative cerebral blood flow (rCBF) in the MCA using a laser Doppler flowmeter. MCAO will be confirmed when the rCBF is maintained at < 20% of the resting condition values during the entire ischemic period.
- Fix the inserted filament to the blood vessel for 2 h while the cerebral artery is occluded, and then carefully withdraw the filament to restore the blood flow for 22 h of reperfusion. Suture the skin by sewing at five places (3-0 silk suture, two half-hitch knots) and allow each mouse to awaken from the anesthesia.