The overall goal of the hypoxic-ischemic neonatal brain injury surgery, followed by cognitive and motor testing, is to have a mouse model to study such injuries and related brain damage. This method can help answer key questions about neonatal hypoxic-ischemic brain injury, such as how such injuries affect motor and cognitive functions. The main advantage of this technique is that it is well-known and is commonly used.
It is also a preclinical model for cerebral palsy and neonatal strokes. Generally, individuals new to this method will struggle because the unilateral right carotid artery is difficult to identify. Demonstrating the procedure, along with myself, will be Yoon-Kyum Shin, a graduate student from our laboratory.
After anesthetizing at least five P-seven 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-degree 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 semi-transparent 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.
When the mice are six weeks old, evaluate their memory based on the learning and avoidance of an adversive stimulus. Use a commercial two-compartment, step-through passive avoidance apparatus for this task. To begin, place a mouse into the bright compartment of the box for 30 seconds.
Then, open the door between the compartments, and record the animal's latency to enter the dark compartment. Allow up to 300 seconds to perform the task. When all four limbs of the mouse are fully inside the dark compartment, close the door.
Then, administer a 1/2 milliamp electric foot shock for two seconds, and return the mouse to its cage. 24 hours later, repeat the test. This time, allow the mouse a full 300 seconds to move into the dark chamber, and do not shock the mouse.
Perform the ladder walking test on the six-week-old mice to discriminate between subtle disturbances of motor function, using both qualitative and quantitative analyses. First, set up the ladder apparatus, and then set up a video camera to record the limb movements of a mouse on the apparatus. Next, place a mouse on the start panel, and start making a recording with the video camera.
Make certain that the video shows the legs in good focus. When the mouse touches the last panel of the ladder apparatus, stop the recording. Then, move the mouse back to the starting panel, and record the mouse crossing the apparatus again.
Do this a total of four times per mouse on each trial date. Later, when analyzing the video, manually count the number of slips made by each limb while playing the video at 1/10 speed. When a limb slides between the bars, this constitutes a slip.
When the mice are six weeks old, assess their grip strength. Use a push/pull strain gauge, outfitted to a two-millimeter diameter triangular wire, all fixed to a panel. Position the mouse on the panel, holding it by its tail.
Then, allow the mouse to just reach the metal wire with its forelimbs. As soon as mice grip the wire, gently pull its tail until it releases its grasp. The apparatus will register the peak force on the wire in grams.
Repeat this test five times before giving the mouse a rest. During the grip strength test, there are noticeable movements of accurate performances. Try to find those movements over five efforts before resting the mouse, for at least one minute to reduce its muscle fatigue.
After the mouse rests for at least one minute, repeat the five-trial series again. Then, give the mouse another rest, and perform the series one last time for that test day. From the data, use the average of the three largest scores for either forelimb or for both forelimbs for analysis.
After harvesting the brains of 14-week-old hypoxia-ischemia injured animals, the brains were evaluated morphologically as having no cortical injury, a mild injury, or a severe injury. The same animals were scanned by an MRI a week earlier, which was also very revealing. Even the morphologically normal brain showed mild injury in the hippocampus.
At six weeks of age, five weeks post-injury, the mice were assessed with various behavioral tasks. Hypoxia-ischemia injured mice were cognitively impaired on the 24-hour passive avoidance task. Because only the right hemisphere was damaged, the mice showed hemiplegic motor functions.
This was revealed by the difference in the percentage of slips on the transverse rungs of the ladder for each forelimb. Moreover, since grip strength involves the motor cortex in the brain, the normal and cortical injury groups showed differences in grip power. No difference was observed between the normal controls and categorically uninjured mice, but the injured mice were clearly impaired.
After watching this video, you should have a good understanding of how to ligate the unilateral right carotid artery to generate a neonatal hypoxic-ischemic mice and how to perform follow-up behavioral analysis. It is important to note that even when there is no visible damage seen on the cortical brain seven days after injury, an MRI may reveal a hippocampal injury. Also, it is important to remember to treat the pups gently and complete the surgery as quickly as possible.
Pups have very weak and thin skins, so be very careful.
