The overall goal of these procedures is to determine muscle functionality by means of several non detrimental functional tests. Strength of the four limbs can be assessed by means of the grip strength test, balance coordination, and muscle condition can be assessed in the two limb and four limb hanging tests. The rotor rod is also used to assess balance, coordination and condition.
Forced treadmill running can either be used to exacerbate disease progression or prior to a functional test to exhaust the mice. These functional tests can be used to reliably assess the role of genetics and the effect of potential therapeutic compounds on muscle performance. The first test to be shown is the four limb grips strength test.
Ensure that the force transducer setting is on peak tension mode for pulling and use grams as the unit reset the meter at the start of each recording. Let each mouse pull three times in a row. Grab the mouse by the tail and move it horizontally toward the grid.
Check that the mouse grasps the grid tightly with both four paws and pull to break its grasp. The highest force applied to the grid will be shown on the transducers display, which can be either manually or automatically recorded. Reject any trials in which the mouse uses only one four PA or the hind limbs or turn during the pole or clearly did not show resistance to the pole.
After the mouse pulled three times, return it to the cage for a resting period of at least one minute. Then let the mouse perform four series of polls each followed by a short resting period. In this way, the mouse pulls a total of 15 times.
The apparatus for the two limb hanging test is a two millimeter thick metal clothes hanger tightly secured to a shelf with tape. Maintain the hangar 37 centimeters above a layer of bedding, holding the mouse via the tail. Bring it near the wire.
Let the mouse grasp the wire with the two four paws only and lower the hind limbs in such a way that the mouse only hangs with the two four paws on the wire. As soon as the mouse is released, start the timer. After being released, a strong mouse will try to catch the wire with all four limbs and the tail, which is allowed.
When a mouse shows improper behavior like balancing on the wire or deliberately jumping off the wire, immediately replace the mouse on the wire without stopping the timer. When mice are able to hang for 600 seconds, take them off the wire and return them to the cage. When mice fall off the wire, stop the timer and record the hanging time directly.
Retest these mice for a maximum of two more times. For the four limb hanging test position the lid of a rat cage 25 centimeters above soft bedding to prevent the mice from harming themselves upon falling, but also to discourage mice from intentionally jumping off the grid. Next, place the mouse on the grid so that it grasp the grid with its four paws.
Then invert the grid so that the mouse is hanging and immediately start the timer. If a mouse is able to hang for 600 seconds, return it to its cage directly. Retest any mice that fall off the grid before 600 seconds for a maximum of two more times for the rotor rod.
Test, set the initial speed at five RPM. Then place up to five mice on the tube as it rotates slowly. Once all of the mice are positioned, increase the speed of the tube from five to 45 RPM over 15 seconds.
Then leave it at 45 RPM for the remainder of the run. Continue to monitor the run throughout the test. The running time is continuously recorded by the software and stops automatically when a mouse falls off the tube.
As this activates the time bar positioned below the tube. Reposition mice that turn around while running without stopping the rotating tube. End the test session for any mice that are able to run 500 seconds and return these mice to their cage.
Give mice that fall earlier. A maximum of two more tries to allow them to improve their running time. For the treadmill exercise test, place the mice on a horizontal treadmill.
Start the treadmill at a running speed of 12 meters per minute. During the first session, encourage the mice to run by gently pushing them when they're near the end of the belt. After the mice have run for 30 minutes, either immediately test them on the two limb hanging test or place them back into their cage.
The four limb grip strength of nine week old female MDX mice was impaired compared to wild type mice as shown here. However, the average fatigue was less than 10%and did not vary between the strains in the two limb hanging Test performed once weekly. Both male MDX and wild type mice displayed a learning curve in the first few weeks of testing.
However, the performance of the MDX mice was worse at all time points compared to the wild type mice. The maximum allowed hanging time of 600 seconds is indicated by the dotted line.Four. Limb hanging performance was assessed once weekly in male MDX and wild type mice.
Over time the MDX mice hung for shorter times than the wild type mice. The rotor rod running times did not differ between young male MDX and wild type mice directly after running on a treadmill for 30 minutes. Female mice were tested with the two limb hanging test while all wild type mice remained hanging until the maximum allowed time.
All MDX mice fell off the wire earlier. The presence of membrane damage was determined by assessing serum creatine kinase levels that leak out of muscle fibers through tears in the membrane. Creatine kinase levels were elevated in MDX mice compared to wild type mice before exercise and treadmill exercise increased levels in MDX mice while they remain low in wild type mice.
Muscles of MDX mice are very vulnerable to treadmill exercise. Worsening disease pathology extensively after a few weeks of running these h and e stainings of the quadriceps of 16 week old non-exercise and treadmill exercised. MDX mice show that extensive fibrosis and necrosis developed the muscles of wild type mice undergoing the same running protocol were not affected.
Differences in genetic background influence functional performance, the two limb hanging test performance significantly differed between two strains of MDX mice. The four limb hanging test results were slightly higher in the mixed background. MDX mice.
The rotor rod running times also slightly differed between the strains. While attempting this procedure, it's important to remember to always use sufficiently powered groups of age and gender matched mice with corresponding genetic backgrounds. Also keep the experimental conditions as standardized as possible.
For example, use the same time of day and weekday, the same room, and the same experimenter.
