A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

The overall goal of this procedure is to quickly and reliably evaluate the neuromuscular function of SOD one G 93 a mice by daily assessment of their hind limb function using a simple phenotypic neurological scoring system. This is accomplished by first observing the hind limbs of the mice while suspending them by the tail. The second step is to allow the mice to walk while observing their gait.

Next, the mice are placed on their sides to test for the presence or absence of the writing reflex. The final step is to perform the data analysis by fitting neurological score data of mouse cohorts into an ordinal logistic regression model. Ultimately, our phenotypic neurological scoring system is used to assess disease progression in the SOD one G 93, a mouse model of a LS for the purpose of discerning the efficacy of therapeutic interventions.

The main advantage of this method over all other existing techniques is that it is quick, simple, reliable, and minimally stressful for the mice. The implications of this method extend to a therapy of neotropic lateral sclerosis or a LS because it allows for the quick assessment of therapeutic interventions in the most common mouse model of the disease. Visual demonstration is critical as the neurological scoring method is difficult to learn because the neurological differences can be subtle.

Begin by housing male SOD one G 93 A mice so that there is one mouse per cage house, female SOD one G 93 A mice up to two per cage. Provide environmental enrichment for the mice in the form of plastic huts. Provide the mice with food and water at Lido record.

Body weights for all mice and studies at the same time. Daily body weight is a sensitive indicator of disease progression and of any malaise that might result from chronic drug treatment. During the study, assess the neurologic score of each mouse daily through a regimen of three tests performed sequentially.

Watch carefully as delineating scores at phenotypic transition points is very difficult In the first test. The tail suspension test, hold the mouse approximately 1.5 inches from the base of the tail over the wire top of the home cage. Observe the function of the hind limbs for a period of one to two seconds and repeat the test three times.

For the walking test. Place the mouse on a clean surface that provides some traction and has a walking distance of at least 25 centimeters. Allow the mouse to walk up to 75 centimeters while observing its ga.

For the writing reflex test, place the mouse on its side and use a stopwatch to measure the time it takes to write itself unassisted from both sides. Only one trial of this test is required. Score each hind limb left or right independently on a scale from zero to four based on the collective outcome of the three phenotypic observation tests.

A sinus score of zero indicating the mouse is presymptomatic. When during the suspension test, the hind limbs are fully extended away from the lateral midline and stay in this position for two seconds or longer. Also, during the walking test, a normal gait is observed.

Assign a score of one indicating the appearance of first symptoms. If during the suspension test, the hind limb is either collapsed towards the lateral midline, partially collapsed towards the lateral midline trembling or abnormally splayed in some other fashion. In addition to the suspension test symptoms just shown, the gait of a mouse that should receive a score of one should still appear normal or slightly lower.

Assign a score of two indicating the onset of Parsis. If during the suspension test, the hind limb is partially or completely collapsed, and in addition during the walking test, the hind limbs are used for forward motion. However, the toes either curl downwards at least twice during a 75 centimeter walk or any part of the foot drags to get a score of two.

The mouse must also still be able to write itself after being placed on both of its sides within 10 seconds. A sinus score of three indicating paralysis. If during the suspension test the mouse shows rigid paralysis in the hind limb or has minimal joint movement, the mouse may still show forward motion during the walking test, however, not by using its hind limbs.

In addition to a sinus score of three, the mouse must still be able to write itself after being placed on both of its sides within 10 seconds. Finally, a of score of four, defining the humane endpoint whenever the mouse shows rigid paralysis during the suspension. Test shows no forward motion during the walking test using the hind limbs, and is also unable to write itself within 10 seconds after being placed on either side.

Male mice typically have a more aggressive disease progression than female mice, as evidenced by a greater proportion of their lifespan displaying symptoms that can be classified with a neurological score of one compared to females. As symptoms increase into the range of a neurological score of two or three, the number of days spent at each score is equal in frequency across genders. Shown here are ordinal logistics, statistical analysis graphs generated using the neuroco protocol that was just described.

These data are from a drug efficacy pharmacology study in which a cohort of SOD one G 93 a mice receiving the test compound TDI 1 73 is compared to a cohort of mice receiving vehicle. No statistically significant differences were observed when genders were separately analyzed, but statistical significance was reached when genders were merged. Though showing similar trends, Kaplan Meyer analysis of survival was not sensitive enough to reveal significant differences even when genders were merged.

Once mastered, the scoring system can be applied in less than a minute per mouse if it is properly performed. After watching this video, you should have a good understanding of how to assess the disease progression in the SOD one G 93, a mouse model of a LS by using the Neuroco system.