This study outlines a surgical model to induce hypoxic-ischemic brain injury in mouse pups, simulating conditions that lead to neuronal damage. The methodology includes ligation of the carotid artery and subsequent exposure to hypoxic conditions.
Secure an anesthetized mouse pup abdomen-up under a dissection microscope.
Make an incision in the neck.
Retract the adipose tissue to expose the unilateral right carotid artery.
Ligate the artery to restrict blood flow to the right brain hemisphere, simulating an ischemic condition.
Close the incision and transfer the pup to a hypoxic chamber.
Once the pup regains consciousness, close the chamber and lower the oxygen levels to establish a hypoxic condition.
Decreased blood supply and oxygen levels in the brain result in excitatory neurotransmitter release.
These neurotransmitters induce neuronal hyperexcitability, calcium influx, and calcium-dependent damage to neuronal membranes, cellular proteins, and DNA, leading to neuronal death.
In response, microglia become activated and release pro-inflammatory cytokines, worsening the brain damage.
Allow the pup to recover and grow for a week.
Anesthetize the pup, position it abdomen-down, and make a scalp incision.
Visualize the brain lesion in the right hemisphere through the semi-transparent skull to detect the hypoxic-ischemic injury.
After anesthetizing at least five P7 mouse pups, position a pup under a dissection microscope with the abdomen up, then clean the neck with alcohol. To begin the surgery, use scissors to make a 0.7-millimeter incision into the neck. Then, carefully remove the adipose tissue using forceps until the unilateral right carotid artery is exposed. Next, using a 5-0 suture, ligate the unilateral right carotid artery.
Now, suture the incision in the neck with 5-0 nylon, and sterilize the incision. Then, allow the pup to recover in a 37 degrees Celsius hypoxic chamber with the lid open for about an hour. Place another surgically manipulated mouse into the hypoxic chamber to recover. Once the pups are all fully awake, close the chamber lid and decrease the gas levels to establish hypoxic conditions.
After 90 minutes of hypoxia, return the brain-injured pups to their cages. A week later, prepare the pups for surgery as before to look for evidence of the injury. Make an incision in the scalp to identify the brain lesion in the posterolateral area of the right hemisphere. Look through the semitransparent skull for evidence of a cortical injury and a hippocampal injury, thus categorize the pups as injured or uninjured controls. After checking, close the scalp with suture and sterilize the incision.
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