Academic Background

Duchenne Muscular Dystrophy (DMD) is a genetic muscular disorder and the most common type of muscular dystrophy in Japan. DMD is caused by a mutation in the dystrophin gene on the X chromosome, leading to the absence or defect of the dystrophin protein in muscle fibers. This results in a cascade of events, including increased muscle fiber membrane permeability, calcium influx, reactive oxygen species (ROS) production, and cellular necrosis. Chronic muscle degeneration leads to the persistent accumulation of inflammatory cells, further exacerbating disease progression. Currently, the diagnosis of DMD relies on physical examination, family history, and laboratory tests, with molecular diagnostic techniques such as microarrays and muscle biopsies also being widely used. However, the pathophysiological mechanisms of DMD are complex, involving inflammation, mitochondrial dysfunction, and redox state dysregulation. Therefore, non-invasively assessing local inflammation and redox status in DMD patients has become an important research focus.

Magnetic Resonance Imaging (MRI) is a crucial tool for evaluating the progression of muscle diseases. Traditional T2-weighted MRI can assess inflammation and muscular dystrophy based on increased muscle water content. However, the redox status in DMD has not been non-invasively evaluated during disease progression. Dynamic Nuclear Polarization MRI (DNP-MRI) is an emerging non-invasive imaging technique that can assess tissue redox status by monitoring the distribution of redox probes. This study aims to use DNP-MRI to evaluate the redox status in the skeletal muscle of DMD model mice (mdx mice) and explore its application in assessing local inflammation.

Source of the Paper

This paper was co-authored by Hinako Eto, Masaharu Murata, Takahito Kawano, Yoko Tachibana, Abdelazim Elsayed Elhelaly, Yoshifumi Noda, Hiroki Kato, Masayuki Matsuo, and Fuminori Hyodo, affiliated with institutions such as Kyushu University, Gifu University, and Suez Canal University. The paper was published in npj Imaging in 2024, titled Evaluation of the Redox Alteration in Duchenne Muscular Dystrophy Model Mice Using In Vivo DNP-MRI.

Research Process and Results

Research Process

1. DNP-MRI Imaging
   The study first used DNP-MRI to perform redox imaging on the skeletal muscle of mdx mice. The experiment selected mdx mice at 5, 9, and 12 weeks of age, along with normal mice as controls. The redox probe carbamoyl-proxyl (CMP) was intramuscularly injected, and DNP-MRI images were acquired at different time points (2, 4, 7, 10, and 13 minutes). The redox status of the skeletal muscle was assessed by analyzing the decay rate of CMP radicals.

2. Evaluation of Vascular Absorption of the CMP Probe
   To verify whether the decay rate of the CMP probe was affected by vascular absorption, the study compared the plasma concentrations of the CMP probe in mdx and normal mice 10 minutes after injection. The results showed no significant difference in plasma CMP concentrations between the two groups, indicating that the decay rate of the CMP probe was primarily influenced by redox reactions rather than vascular absorption.

3. Pathological Evaluation
   The study evaluated muscle injury markers (such as glutamic oxaloacetic transaminase GOT, creatine phosphokinase CPK, and lactate dehydrogenase LDH) in mdx mice through blood biochemical tests. The results showed that the concentrations of these markers were significantly higher in mdx mice than in normal mice, indicating muscle fiber damage. Additionally, histopathological examinations (such as hematoxylin and eosin staining and Masson’s trichrome staining) revealed focal necrosis and regeneration of muscle fibers in mdx mice, accompanied by inflammatory cell infiltration.

4. Mitochondrial Function Evaluation
   The enzymatic activities of mitochondria in muscle fibers were assessed using succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) staining. The results showed that necrotic muscle fibers in mdx mice were negative for SDH staining but positive for COX staining, indicating impaired mitochondrial function.

5. Reactive Oxygen Species (ROS) Generation Evaluation
   The infiltration of mature macrophages in the skeletal muscle of mdx mice was detected using immunohistochemistry, and ROS generation was assessed using dihydroethidium (DHE) fluorescence staining. The results showed a significant increase in macrophage infiltration and ROS generation in the necrotic areas of muscle fibers in mdx mice.

6. In Vitro Evaluation of CMP Radical Reduction Reaction
   The study further evaluated the effects of macrophage numbers and mitochondrial concentration on the reduction rate of CMP radicals in vitro. The results showed that the reduction rate of CMP radicals increased linearly with both macrophage numbers and mitochondrial concentration.

Main Results

1. DNP-MRI Imaging Results
   DNP-MRI imaging showed that the decay rate of CMP radicals in mdx mice was significantly higher than in normal mice, indicating altered redox status in the skeletal muscle of mdx mice. This result is consistent with the pathological features of muscle inflammation in mdx mice.

2. Pathological Evaluation Results
   Both blood biochemical tests and histopathological examinations revealed significant muscle fiber damage and regeneration in mdx mice, accompanied by inflammatory cell infiltration. Mitochondrial function evaluation further confirmed impaired mitochondrial function in the muscle fibers of mdx mice.

3. ROS Generation Evaluation Results
   Immunofluorescence staining showed a significant increase in macrophage infiltration and ROS generation in the necrotic areas of muscle fibers in mdx mice, indicating that inflammatory responses play an important role in muscle damage in mdx mice.

4. In Vitro Experimental Results
   In vitro experiments demonstrated that both increased macrophage numbers and mitochondrial concentration accelerated the reduction reaction of CMP radicals. However, in mdx mice, despite reduced mitochondrial concentration, the reduction rate of CMP radicals still significantly increased due to macrophage infiltration and ROS generation.

Conclusions and Significance

This study used DNP-MRI to non-invasively evaluate the redox status in the skeletal muscle of DMD model mice (mdx mice) and found that the decay rate of CMP radicals in mdx mice was significantly higher than in normal mice. This result indicates that DNP-MRI can effectively assess local inflammation and redox status in DMD model mice. Additionally, the study revealed that macrophage infiltration and ROS generation play a crucial role in muscle damage in mdx mice, while impaired mitochondrial function further exacerbates redox state dysregulation.

The scientific value of this study lies in its pioneering use of DNP-MRI to non-invasively evaluate the redox status in DMD model mice, providing a new tool for studying the pathological mechanisms of DMD. Furthermore, the results suggest that DNP-MRI can be used to assess local inflammation and redox status in DMD patients, offering important insights for early diagnosis and treatment strategies.

Research Highlights

1. Non-Invasive Imaging Technique: This study is the first to use DNP-MRI to non-invasively evaluate the redox status in DMD model mice, providing a new tool for studying the pathological mechanisms of DMD.
2. Local Inflammation Assessment: The results demonstrate that DNP-MRI can effectively assess local inflammation and redox status in DMD model mice, offering important insights for early diagnosis and treatment strategies.
3. Role of Macrophages and ROS: The study found that macrophage infiltration and ROS generation play a crucial role in muscle damage in mdx mice, further elucidating the pathological mechanisms of DMD.

Other Valuable Information

The study also explored the role of mitochondrial function in the reduction reaction of CMP radicals, finding that despite impaired mitochondrial function in mdx mice, the reduction rate of CMP radicals still significantly increased due to macrophage infiltration and ROS generation. This finding provides new directions for further research into the pathological mechanisms of DMD.