A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We have developed a composite phenotype scoring system for the evaluation of disease severity and progression. In mouse models of cerebellar ataxia, we perform four different tests, the ledge test, hind limb, clasping, gait, and kyphosis, using a scale of zero to three for each test for a combined score of zero to 12. For all four measures, the ledge test has performed to directly measure coordination.

Hind limb clasping is a marker of disease progression in a number of mouse models of neuro degeneration. Gait is a measure of coordination and muscle function. Kyphosis is curvature of the spine in the cervical thoracic region, common in mouse models of motor system and spinal cerebellar neurodegeneration who perform each test and record the score.

The average sum of the four scores or the average composite phenotype score is calculated for each mouse group and plotted. When applying this protocol to different mouse models, it is important to first analyze the scores of the individual tests to determine the ideal combination of the four described measures. This specific protocol was designed to discriminate between affected and non-affected individuals, while also quantifying the progression of disease phenotype in mouse models of spinal cerebellar ataxia.

Type seven. Hi, I'm Stephanie Fur from the laboratory of Dr.Gwen Garden in the Department of Neurology at the University of Washington. Today we will show you a simple and sensitive method for evaluating disease severity in mouse models of cerebellar ataxia.

We use this procedure in our laboratory to quantify phenotypic differences in mouse models of spinal cerebellar ataxia type seven or SCA seven, and to follow the progression of the SCA seven phenotype over time. So let's get started. The ledge test is a direct measure of coordination, which is impaired in cerebellar, ataxias and many other neurodegenerative disorders.

This measures the most directly comparable to human signs of cerebellar ataxia to prevent bias. The experimenter performing the assessments should not have knowledge of the animal's genotype. During assessments, individual measures are scored on a scale of zero to three, where zero represents the absence of the phenotype, and three represents its most severe manifestation.

Begin by lifting the mouse from the cage and placing it on the cage's ledge. Observe the mouse as it walks along the cage ledge and lowers itself into its cage. A wild type mouse will walk along the ledge without losing its balance and will lower itself back into the cage gracefully using its paws.

This behavior is assigned a score of zero. If the mouse loses its footing while walking along the ledge, but otherwise appears coordinated, it receives a score of one. If it does not effectively use hind legs or cannot gracefully lower itself into the cage.

It receives a score of two if it falls off the ledge or nearly so while walking or attempting to lower itself or shakes and refuses to move at all. Despite encouragement, it receives a score of three. Conduct the ledge test three times to ensure an accurate measurement and record the ledge test score.

Now let's see how to score hind limb clasping. Hind limb clasping is a marker of disease progression in a number of mouse models of neurodegeneration, including certain cerebellar ataxias. To begin the assessment, grasp the tail near its base and lift the most clear of all surrounding objects.

Observe the hind limb position for approximately 10 seconds. If the hind limbs are consistently splayed outward away from the abdomen, it is assigned a score of zero. If one hind limb is retracted toward the abdomen for more than 50%of the time suspended, it receives a score of one.

If both hind limbs are partially retracted toward the abdomen for more than 50%of the time suspended, it receives a score of two. If the hind limbs are entirely retracted and touching the abdomen for more than 50%of the time suspended, it receives a score of three. Conduct the test three times and record the hind limb clasping score.

Now let's see how to assess gait. The gait test is a measure of coordination and muscle function. To begin the test, remove the mouse from its cage and place it on a flat surface with its head, head facing away from the investigator.

Observe the mouse from behind as it walks. If the mouse moves normally with its body weight supported on all limbs with its abdomen not touching the ground, and with both hind limbs participating evenly, it receives a score of zero. If it shows a tremor or appears to limp while walking, or its feet are slightly pointed away from its body, it receives a score of one.

If it shows a severe tremor, severe limp, lowered pelvis or the feet point away more severely from the body during locomotion, it is assigned.Two. If the mouse has difficulty moving forward and drags its abdomen along the ground, it receives a three. Observe the mouses GA three times and recorded skate score.

Now let's see. The kyphosis test kyphosis is a characteristic dorsal curvature of the spine. That is a common manifestation of neurodegenerative disease involving motor systems, including spinal cerebellar ataxias in mouse models.

It is caused by a loss of muscle tone in the spinal muscles secondary to neurodegeneration. To begin, remove the mouse from its cage and place it on a flat surface. Observe it as it walks.

If the mouse is able to easily straighten its spine as it walks and does not have persistent kyphosis, it receives a score of zero. If the mouse exhibits mild kyphosis but is able to straighten its spine, it receives a score of one. If it is unable to straighten, its completely and maintains persistent, but mild kyphosis, it receives a score of two.

If the mouse maintains pronounced kyphosis as it walks or while it sits, it is assigned a score of three. Observe the mouse's kyphosis score three times. Place the mouse back into its cage and record its kyphosis score.

Here is a representative graph of a composite phenotype assessment of a murn transgenic spino, cerebellar ataxia type seven model known as Flocked SCA 7 92 Q.The scores of the four measures were summed for each mouse, and the average composite score was calculated for both genotypes of each age. Here f Fluxed, SCA 7 92 Q mice exhibit A progressive SCA seven phenotype that is significantly different from nont transgenic litter mates. Importantly, this increasing phenotype score over time is consistent with the progressive nature of the human disease.

We've just shown you how to utilize a composite phenotype scoring system to evaluate disease severity and progression in mouse models of cerebellar ataxia. When doing this procedure, it's important to remember that obesity is confounding factor in all four tests described. The results of individual tests may be analyzed separately to first determine their ideal combination in a composite phenotype scoring system, most appropriate for your mouse model.

So that's it. Thanks for watching and good luck with your experiments.