The overall goal of the following experiment is to identify changes to balance performance in a mouse model of aging. This is achieved by first training mice on an apparatus in order to allow mice to acclimate to it, as well as learn the task as a second step. Mice are tested on the apparatus to assess their motor coordination and balance.
Then mice are challenged with the vestibular stimulus and retested on the apparatus. Again, the results will show changes in balance with age based on rotor rod and balance beam data. The main advantage of this technique over existing methods like the vestibular ocular reflex test, which tracks eye movements, is that this is a simple and non-invasive method for measuring balance performance.
Although this method can provide insight into age-related changes to balance performance in mice, it can also be applied to other systems such as ery and neuromuscular disorders. Demonstrating that procedures will be Victoria Tongue, a PhD candidate within my laboratory. Edward Dabney, a research assistant in the lab, and Thomas Burton, the manager of the Bosch Institute Animal Behavioral Facility at the University of Sydney.
In the rotor rod test, mice must walk along rotating and accelerating dowels. When they lose their balance, they trigger a magnetic sensor. Scored parameters are recorded by the rotor rod apparatus.
Begin with installing dowels into each lane of the rotor.Rod. Dowels of different sizes are provided for mice and rats. However, using the rat dowels for mice works well and prevents mice from clinging to the dowels instead of falling.
Next position the magnetic landing platforms on the wire below each lane of the rotor rod. Make sure that the platforms are not tilted to touch the floor and that they are as close as possible to the right side magnetic wall without touching it. Finally, slide two.
Clear plastic panels in front of each lane of the rotor rod. The shorter panel is placed below and the longer panel is placed above. Bring the mouse cages into the testing room 10 minutes before starting the trials while the mice acclimate.
Input the test parameters for the accelerating rotor rod test. First, set the maximum duration of the test to 60 seconds. Next, set the number of lanes to be used or the number of mice to be tested.
Then set the speeds. Set the test to start at five RPM and set the maximum to 44 RPM. Have the speed ramp up over 60 seconds.
Lastly, set the dowel parameters for the use of rat dowels rotating in a forward direction. When the mice are ready, place the first group to be tested on the stationary dowels for five minutes, allowing them to acclimate. Then gently nudge the mice to face the back of the rotor rod and start the test when all the mice are facing the right direction.
Erection, after the mice have fallen, return them to their cages for 10 minutes with free access to food and water between trials while the mice rest, clean off the animal waste from the dowels lanes and landing platforms. Also reset the landing platforms for the next test. Repeat the process for a total of eight trials per mouse.
The first three to five trials are used as training trials. After all eight trials, thoroughly clean the dowels lanes and landing platforms of the rotor rod with 70%ethanol and clean the clear plastic panels with a mild detergent. In this test, mice walk up a one meter long, 14 millimeter diameter exposed beam along a slight slope to their goal.
A dark chamber containing the subject's housing dome Two cameras are positioned on either side of the balance beam. They're each angled to capture the entire length of the beam. The video recordings document the trials below the beam.
The floor is covered with cushioning material. Mice that fall must be picked up immediately and placed inside the goal box to recover. The interior of the goal box is lined with a paper towel to easily remove animal waste between tests.
The box is large enough that a housing dome can be fit within it. Before testing a mouse, place it in the goal box for two minutes. If a mouse tries to leave the goal box, cover the opening to the beam for five seconds with a gloved hand to discourage it from leaving again.
Next, the mouse must be trained for the task. Place it on the beam just outside of the goal box and allow it to walk up to the goal box. Then let it rest for a minute, then place it further down the beam and let it walk up again.
Always give the mouse a minute In the goal box between training trials. Keep the mouse further and further down the beam until it walks up from the starting line without assistance. Now to conduct a test, start the recording and then place the mouse on the start line.
Stop the recording when the mouse reaches the box or when the mouse falls off the beam between trials. Let the mouse rest for a minute in the goal box, and in the meantime, clean off any urine or feces from the beam and underlying foam. Each mouse should be tested five times in this manner.
To add an additional vestibular challenge to this test, apply the rotator first, acclimate the mice to the sound of the rotator at three hertz. Operate it for 20 seconds. Then transfer the mouse to the chamber on the rotator.
If you are having trouble lowering the mouse into the chamber, give the mouse an object to grip onto before lowering both into the chamber. Once the mouse is in the chamber, remove the object, Replace the lid and run the rotator for 20 seconds. At three hertz, start the video recordings as it is spinning.
After 20 seconds, turn off the rotator and manually stop the wheel as quickly as possible. Then immediately move the mouse to the start line and allow it to complete the trial. As usual, this test can be performed one to three times before the mice stop cooperating.
Never forget to clean the balance beam and change the paper towel between mice for the rotor rod. Test the time to fall for one. One month old and one nine month old mouse increased steadily during trials one to five and were therefore considered training trials.
The data from where score is stabilized shown by a dashed line was used for data analysis. One month old mice were able to stay on the rotating dowel significantly longer than nine month old mice. The data from a small sample size is represented as the mean plus or minus one standard deviation.
The impact of vestibular stimulation on balance be performance was also greater with age. A 22nd spin at three hertz prior to the trial increase the time taken to traverse the balance beam between the start and finish lines in nine and 13 month old mice, but not in young mice. Significance was assessed using repeated measures in Nova with a TUI post hoc test.
When performing this task, it is important to remember to transfer the animal from the vestibular challenge to the apparatus as soon as possible to minimize the chance of the animal recovering from the stimulus before completing the task Following this procedure. Other techniques like PCR Calcium imaging and patch clamp electrophysiology can be used to answer additional questions like whether changes in motor and balance performance can be correlated with changes in vestibular hair cell function.
