Assessing Functional Performance in the Mdx Mouse Model
March 27th, 2014
The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.
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.
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Overview
This article discusses the importance of assessing muscle functionality in mouse models for neuromuscular disorders. Several non-detrimental functional tests are described to evaluate muscle performance and the effects of therapeutic compounds.
Key Study Components
Area of Science
- Neuromuscular disorders
- Muscle functionality assessment
- Preclinical testing in mouse models
Background
- Improved muscle function is a primary outcome in clinical trials for neuromuscular disorders.
- Preclinical assessments are crucial for evaluating potential therapies.
- Functional tests can provide insights into muscle performance.
- Mouse models are commonly used for these assessments.
Purpose of Study
- To determine muscle functionality using various tests.
- To assess the impact of genetics and therapeutic compounds on muscle performance.
- To establish reliable methods for evaluating muscle strength and coordination.
Methods Used
- Grip strength test for assessing limb strength.
- Two limb and four limb hanging tests for muscle condition evaluation.
- Rotor rod test for balance and coordination assessment.
- Forced treadmill running to evaluate disease progression and muscle performance.
Main Results
- Functional tests reliably assess muscle performance.
- Grip strength test is highlighted as a key assessment tool.
- Tests can indicate the effects of therapeutic interventions.
- Methods can be adapted to evaluate various genetic backgrounds.
Conclusions
- Functional tests are essential for preclinical evaluation of therapies.
- These assessments can guide future clinical trials.
- Mouse models provide valuable insights into neuromuscular disorders.
Frequently Asked Questions
Executive Industry Relevance
Reliable functional assessment in the mdx mouse model is critical for preclinical evaluation of candidate therapies targeting Duchenne muscular dystrophy. Standardized, reproducible muscle performance tests enable robust comparison of therapeutic effects and genetic influences across studies. These validated endpoints support predictive confidence and risk-adjusted advancement in neuromuscular drug discovery pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables quantitative interrogation of muscle function to clarify therapeutic hypotheses.
- Supports biological de-risking by distinguishing genetic and treatment effects on muscle performance.
- Facilitates functional target validation through standardized, reproducible endpoints.
Screening & Assay Development
- Prepares validated in vivo systems for downstream compound screening workflows.
- Ensures assay reproducibility and comparability across laboratories via standard operating procedures.
- Delivers quantitative outputs for reliable evaluation of candidate interventions.
Translational & Preclinical Research
- Aligns preclinical endpoints with clinically relevant measures of muscle function.
- Enables continuity from discovery through preclinical validation by tracking disease progression and intervention effects.
- Supports risk-adjusted decisions for advancing therapeutic candidates based on functional outcomes.
Pipeline & Workflow Integration
These functional tests position within the preclinical continuum, bridging early discovery, lead identification, and translational research for neuromuscular disorders.
- Discovery Biology: Provides hypothesis-driven assessment of muscle strength, coordination, and disease progression.
- Screening: Offers reproducible, quantitative readouts for evaluating intervention efficacy.
- Analytics: Enables statistical comparison of functional performance across genotypes and treatments.
- Translational Research: Connects preclinical muscle function data to clinical trial endpoints.
- Enterprise Reuse: Establishes a standardized platform for ongoing neuromuscular research and therapeutic evaluation.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Promotes standardization, reproducibility, and scalability of functional assessments.
- Strategic Value: Informs go/no-go decisions and enhances capital efficiency by prioritizing candidates with robust functional benefit.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of neuromuscular therapeutic programs.
Implementation Considerations
- Requires expertise in animal handling and functional test execution.
- Needs calibrated instrumentation for grip strength, rotarod, and treadmill assays.
- Demands rigorous cross-team standardization of protocols and data collection.
- Must account for genetic background, age, and gender in experimental design.
- Dependent on consistent environmental and procedural conditions to minimize variability.
