J. Biochem, 1995, Vol. 118, No. 5 959-963
© 1995 Japanese Biochemical Society
Visualization of Dystrophic Muscle Fibers in Mdx Mouse by Vital Staining with Evans Blue: Evidence of Apoptosis in Dystrophin-Deficient Muscle1
* Department of Biology, The University of Tokyo 3–8–1 Komaba, Meguro-ku, Tokyo 153
Department of Physiology, Saitama Medical College Moroyama-cho, Iruma-gun, Saitama 350–04
Pharmaceuticals Laboratory II, Mitsubishi Kagaku Corporation, Research Center, 1000 Kamoshida, Midori-ku Yokohama 227
2 To whom correspondence should be addressed. Phone: +81–3–5454–6637, Fax: +81-3-3485-2904
Received June 8, 1995;
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Abstract |
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Degenerating muscle fibers in the skeletal muscle of mdx mice were visualized by vital staining with Evans blue. Evans blue injected intravenously stained only degenerating muscle fibers which were visible as blue fibers macroscopically and could also be seen as red fluorescent fibers microscopically. Evans blue-stained muscle fibers were either hypercontracted or degrading. Intact or regenerating muscle fibers in mdx mice and muscle fibers in B10 control mice were not stained with the dye. DNA isolated from Evans blue-stained fibers exhibited fragmentation to approximately 180 base pairs on agarose gel electrophoresis. Such DNA fragmentation was not found in DNA from unstained muscle fibers in mdx or B10 mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL)-positive myonuclei were also found in Evans blue-stained muscle fibers but not in unstained ones. These results indicate that degenerating muscle fibers in the mdx mouse show an increase in membrane permeability and undergo apoptosis. Vital staining with Evans blue is useful not only for distinguishing degenerating muscle fibers, but also for studying the degeneration process biochemically in dystrophin-deficient muscle. This method may also be useful for evaluating the therapeutic effects of drug administration, gene transfer, and myoblast transfer in the mdx mouse.
Duchenne muscular dystrophy, dystrophin, Evans blue, mdx mouse, muscle degeneration
1 A part of this work has appeared in abstract form in Mol. Biol. Cell. 5, 27a, 1994. This study was supported by research grants No. 5A-1 from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan, No. 06213214 for scientific research on priority areas (Biomechanics) and No. 06804054 from the Ministry of Education, Science and Culture of Japan and the Fugaku Trust for Medicinal Research.
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