Assessing the Neuroprotective Effects of a Test Compound in a Middle Cerebral Artery Occlusion Mouse Model

Begin with anesthetized mice with sutures on the external carotid artery, or ECA, and internal carotid artery, or ICA, and a thin filament occluding the middle cerebral artery, or MCA.

Position a laser Doppler probe to measure the relative cerebral blood flow in the MCA. A decrease in blood flow confirms MCA occlusion.

Secure the filament in the ICA to maintain MCA occlusion.

This occlusion restricts oxygenated blood supply to the brain, leading to neuronal death and the formation of dead tissue or an infarct.

After the occlusion period, withdraw the filament to restore blood flow.

Suture the incision and allow the mice to recover.

Divide the mice into two groups and administer a test compound via oral gavage to the treated group, while the control group remains untreated.

The compound enters the circulation, reaches the brain, limits neuronal apoptosis, and inhibits further neuronal death compared to the untreated control group.

A smaller infarct in the treated group compared to the control group confirms the compound's neuroprotective effect.

To set up a mouse middle cerebral artery occlusion model, or MCAO, confirm a lack of response to tale stimulus in an anesthetized six-week-old, 22 to 25-gram male C57 black 6 mouse. And place the mouse on a 36.5 degree Celsius body temperature holding blanket connected to a thermometer.

After removing all of the hair from the chest and neck of the mouse by shaving and depilatory cream, place the mouse under a stereo microscope and make a two centimeters skin incision in the center of the neck. Carefully isolate the left common carotid artery, external carotid artery, and the branch of the internal carotid artery from the surrounding connective tissues. And use 4-0 silk suture to ligate the external carotid artery and the common carotid artery, with a half-hitch knot to temporarily block the blood flow into the internal carotid artery.

Insert a 0.1 to 0.12 millimeter thick, 11 millimeter long, silicone coated, 8-0 monofilament, 4-0 zero silk suture through the internal carotid artery to the origin of the left middle cerebral artery. And use a laser Doppler flow meter to measure the decrease in relative cerebral blood flow artery. Fix the inserted filament to the blood vessel for two hours, while the cerebral artery is occluded.

At the end of the occlusion period, carefully withdraw the filament to restore the blood flow for 22 hours of reperfusion. And use 3-0 silk sutures to suture the skin in five places with two half hitches knots. Then, return the mouse to its cage for anesthetic recovery. One hour after the end of reperfusion, administer 300 milligrams per kilogram body weight G-Rex to the experimental animals only by oral gavage.