ZeEfficacy – Efficacy Services

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Duchenne Muscular Dystrophy (DMD) Model

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to the alterations of a protein called dystrophin that helps keep muscle cells intact. 

Muscle tissue specification, development and function in zebrafish are highly conserved with other vertebrates, including humans. In addition, the impact of disease and therapeutics can be studied much earlier due to its fast development. Transparent and accessible embryos allow for non-invasive tissue analysis, immunohistochemistry and automated locomotion monitoring.

DMD model is designed for fast and high-throughput efficacy evaluation of candidate therapeutic compounds. This model allows identifying compounds that prevent muscle degeneration and the subsequent locomotor alterations.

Applications

  • Study the causes and mechanisms of muscle impairments.
  • High throughput screening of potential therapies for DMD.

Advantages

High conservation of zebrafish muscles with vertebrates and humans.

DMD muscle damage is already evident in embryos, which allows early studies of disease impact, and therefore, earlier drug efficacy testing.

Transparent embryos allow the optical birefringence-based evaluation of muscular integrity.

Automated locomotion monitoring highlights muscle functionality impairment rapidly.

Method description

Early-life Sapje dystrophin-null mutant embryos are exposed to the molecule of interest in order to determine mortality/teratogenicity and muscle integrity dose-effect curves. Muscle integrity is evaluated through optical birefringence, an optic phenomenon resulting from the diffraction of polarized light through the pseudo-crystalline array of the muscle sarcomeres. Zebrafish healthy muscles can be detected as a bright area in an otherwise dark background. Muscle damage, as seen in Sapje mutant, can be detected by a reduction in the birefringence as shown in Figure 1. Phenotypic analysis must be coupled with genotypic homozygous confirmation to determine the percentage of homozygotes that recover the dystrophic phenotype after treatment.

Readouts

  • Toxicity: determination of Lethal Concentration 50 (LC50), Benchmark Dose (BMD) and mortality.
  • Muscle integrity: determined through optical birefringence.
  • Genotyping: individual phenotype-genotype correlations in homozygous, heterozygous and wt larvae.
  • Immunohistochemistry (optional): staining against dystrophin and actin to further characterize muscle tissue integrity.
  • Locomotion activity (optional): traced and analyzed by DanioVision™ software (Noldus IT) automated video monitoring at 6 dpf.
Figure 1. Analysis of muscle integrity through optical birefringence. Characteristic muscular phenotypes of wild-type zefrafish, heterozygous Sapje mutants, homozygous Sapje mutants (dystrophin-null), and homozygous mutants after treatment with an active compound that recovers muscle integrity.
Figure 2. Muscular dystrophy recovery on Sapje (dystrophin-null) mutants after treatment. Percentage of individuals that express DMD muscular damage after treatment with vehicle (negative control), and two active compounds that partially recovers wild-type phenotype. Determined via optical birefringence qualitative scoring of muscular integrity (continuous vs scattered pattern).