Abstract
BackgroundDuchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice.MethodsMdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2FLuc/+DMD−/−) were created to serve as a tool for researchers to evaluate muscle inflammation. Luciferase expression was assayed by immunohistochemistry to insure that it correlated with muscle lesions. The luciferase signal was quantified by optical imaging and luciferase assays to verify that the signal correlated with muscle damage. As proof of principle, Cox2FLuc/+DMD−/− mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment.ResultsIn this investigation, a novel reporter mouse (Cox2FLuc/+DMD−/− mice) was created and validated for non-invasive quantification of muscle inflammation in vivo. In this dystrophic mouse, luciferase is expressed from cyclooxygenase 2 (Cox2) expressing cells and bioluminescence is detected by optical imaging. Bioluminescence is significantly enhanced in damaged muscle of exercised Cox2FLuc/+DMD−/− mice compared to non-exercised Cox2FLuc/+DMD+/+ mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2FLuc/+DMD−/− mice in response to a course of steroid treatment. Reduction in bioluminescence is detectable prior to measurable therapy-elicited improvements in muscle strength, as assessed by traditional means. Biochemical assay of luciferase provides a second means to quantify muscle inflammation.ConclusionsThe Cox2FLuc/+DMD−/− mouse is a novel tool to evaluate the therapeutic benefits of drugs intended to target inflammatory aspects of dystrophic pathology. This mouse model will be a useful adjunct to traditional outcome measures in assessing potential therapeutic compounds.
Highlights
Duchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis
After crossing the Cox2FLuc/+ mouse to the DMD−/− mdx background to create Cox2FLuc/+ DMD−/− mice, we show that dystrophic features of inflammation can be monitored non-invasively by bioluminescent optical imaging
Bioluminescence is detectable in muscles from cardiotoxin-injured Cox2FLuc/+ mice Muscle injury studies were carried out to determine whether the luciferase signal generated by the cyclooxygenase 2 (Cox2) promoter in Cox2FLuc/+ mice was sufficiently strong to be detectable by optical imaging
Summary
Duchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Mdx mice are weaker than age-matched wild-type mice when evaluated by a variety of muscle strength tests [9]. Their serum creatine kinase remains elevated throughout their lives [10]. The mdx mouse has been a highly useful tool for pre-clinical studies to facilitate drug discovery for dystrophinopathies
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