A reporter mouse for optical imaging of inflammation in mdx muscles

BACKGROUND: 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. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice. METHODS: Mdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2(FLuc/+)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, Cox2(FLuc/+)DMD(−/−) mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment. RESULTS: In this investigation, a novel reporter mouse (Cox2(FLuc/+)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 Cox2(FLuc/+)DMD(−/−) mice compared to non-exercised Cox2(FLuc/+)DMD(+/+) mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2(FLuc/+)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. CONCLUSIONS: The Cox2(FLuc/+)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

Long-term administration of the TNF blocking drug Remicade (cV1q) to mdx mice reduces skeletal and cardiac muscle fibrosis, but negatively impacts cardiac function

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in the gene encoding dystrophin (DYS). Tumor necrosis factor (TNF) has been implicated in the pathogenesis since short-term treatment of mdx mice with TNF blocking drugs proved beneficial; however, it is not clear whether long-term treatment will also improve long-term outcomes of fibrosis and cardiac health. In this investigation, short and long-term dosing studies were carried out using the TNF blocking drug Remicade and a variety of outcome measures were assessed. Here we show no demonstrable benefit to muscle strength or morphology with 10mg/kg or 20mg/kg Remicade; however, 3mg/kg produced positive strength benefits. Remicade treatment correlated with reductions in myostatin mRNA in the heart, and concomitant reductions in cardiac and skeletal fibrosis. Surprisingly, although Remicade treated mdx hearts were less fibrotic, reductions in LV mass and ejection fraction were also observed, and these changes coincided with reductions in AKT phosphorylation on threonine 308. Thus, TNF blockade benefits mdx skeletal muscle strength and fibrosis, but negatively impacts AKT activation, leading to deleterious changes to dystrophic heart function. These studies uncover a previously unknown relationship between TNF blockade and alteration of muscle growth signaling pathways