A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

The overall goal of this methodology video is to demonstrate the proper way to perform a battery of neonatal motor tests so that they are easily reproducible and quantifiable. This video will show a series of neonatal motor tests as well as how to perform and score each individual test. One of the problems with other published behavior papers is that the details of these tests do not translate well with just words, and have led to results by different researchers that cannot be compared.

The main advantage of this series of tests is that it uses a standardized, quantifiable scoring system that will allow for better and more accurate reporting of results. The tests we demonstrate here can be used to determine motor deficits, validate an injury or disease model, or determine whether the treatment is working. Crawling develops in the mouse pup within the first five postnatal days.

By day ten, pups transition to walking. Therefore, the ambulation test is performed on day eight. Begin by placing the mouse in a clear enclosure where it is visible from all sides.

Observe the mouse for three minutes and score its ambulation. If the pup does not walk on its own, gently prod the pup’s tail to encourage walking. If there is no movement, give a score of zero.

Give crawling with asymmetric limb movement a score of one. Asymmetric limb movement is when the pup’s paw placement is not consistent and its stepping pattern is irregular. Give slow crawling with symmetric limb movement a score of two.

In this case, the hind paws must meet the forepaws during each step, and each step must smoothly transition to the next. If the mouse is crawling quickly and symmetrically or walking, give this behavior a score of three. During this developmental time period there is a measurable change in the hindlimb posture where the angle between the hindlimbs decreases during the transition from crawling to walking.

To measure the foot angle, video record the pup from either above or below for three minutes. In the video look for segments where the mouse takes a full stride in a straight line with both feet flat on the ground. Measure the foot angle of the pup by drawing a line from the end of the heel to the tip of the longest toe.

Do not take measurements while the pup is stationary or turning. The righting reflex is demonstrated when a mouse pup flips onto its feet from a supine position. This reflex develops in mice between one and 10 days old.

To test for this reflex, place a pup on its back on a bench pad and hold it supine for five seconds, then release the pup and record the time it takes for it to return to a prone position. Also record the direction of righting. Give each pup a maximum of one minute to turn over.

Negative Geotaxis is an innate behavior observable when a pup is placed facing downward and naturally turns around. This reflex develops in rodents between three and 15 days of age. Place the pup with its head pointing downward on a 45 degree incline and hold for five seconds, then release the pup.

Record when, and to which direction, the pup turns, allowing it two minutes to show the behavior. Mice that fall down the incline or fail to turn can be retested, eliminated, or given a score of zero, just be consistent. For mice over 10 days old, use the Front-Limb Suspension Test to detect strength differences between the front limbs.

Hold the pup firmly by the body and allow it to grasp a stiff wire with both forepaws, then release the pup and time how long it takes for it to fall. Record if the pup releases one paw before the other or both paws at the same time. For pups between two and 14 days old use the Hindlimb Suspension Test to detect differences in hindlimb strength and neuromuscular function.

Holding the pup by its tail, gently lower it into a 50 milliliter conical tube with its hindlimbs placed over the rim. Then release the pup and record the time it takes for it to fall. In addition, score its hindlimbs’posture.

Give a score of four if the hindlimbs are separated normally and the tail is raised. If the hindlimbs are closer together but they seldom touch, give the pup a score of three. Score a two if the hindlimbs are close to each other and often touching.

Score a one if the hindlimbs are almost always in a clasped position with the tail raised. And score a zero for constant clasping of the hindlimbs with the tail lowered, or failing to hold onto the tube for any period of time. When a pup attempts to lift its body using its hindlimbs this is considered a pull.

These can be counted and recorded as well. The Grip Strength Test examines the strength of all four paws at the same time. Grip strength can be determined as early as five days and as late as 15 days old.

Place the pup onto a piece of fiberglass screen. Allow the pup to adjust to this environment for about five seconds, then slowly invert the screen to 180 degrees. When the pup falls off, record the angle of the screen.

If the pup is able to hold onto the screen when it is fully inverted, record the latency for it to fall. Use the Grasping Reflex to determine specific paw grasping deficits. This reflex usually appears between three and 15 days.

To begin, grip the pup by the scruff of its neck, the pup should become instinctively immobile and relaxed. Next, stroke the bottom side of each paw with the blunt rounded side of a razor blade and record the presence or absence of grasping. Then using the equation provided in the text calculate the pup’s paw preference.

The Cliff Aversion Test measures the pup’s labyrinth reflexes as well as strength and coordination. The test can be used on pups from postnatal day one to 14. For this test use a box with a flat elevated ledge.

Preset the box with at least five mice. Now place a pup on the edge of the box with its forepaws, digits, and snout suspended over the edge. Then release the pup and start the timer.

Record the time it takes for the mouse to turn away from the edge. Allow at least 30 seconds to complete the test. If a pup falls, it may be retested.

See the text for more details. Cerebral palsy was induced in six day old mouse pups as previously described. Surgical sham pups were used as controls.

Representative results at 48 hours post injury are shown. In the Ambulation Test the cerebral palsy pups ambulate less symmetrically at 48 hours post injury when compared to shams. At 48 hours post injury the hind paw angle was more splayed in the CP mice when compared to sham animals.

CP mice are slightly, but significantly weaker in hindlimb strength when compared to shams. Sham pups have significantly stronger grip strength than CP pups. CP mice grasped with fewer paws when compared to shams.

At 48 hours following injury CP mice show a right hind paw preference. By using this set of neonatal motor tests results from different research groups can be reliably reported and compared. When performing these tests make sure to be consistent.

The mice are young and need rest between trials. In addition they need to be kept warm and returned to the dam occasionally for feeding. Be sure to perform these tests at the same time of day for greater consistency and accuracy of results, also refer to the written text for more specific details, including a distinction between nonparticipation and the inability to perform a task as well as other helpful hints.