VISUALIZATION OF DYSTROPHIC MUSCLE-FIBERS IN MDX MOUSE BY VITAL STAINING WITH EVANS BLUE - EVIDENCE OF APOPTOSIS IN DYSTROPHIN-DEFICIENT MUSCLE

Degenerating muscle fibers in the skeletal muscle of mdx mice were vis ualized by vital staining with Evans blue, Evans blue injected intrave nously stained only degenerating muscle fibers which were visible as b lue fibers macroscopically and could also be seen as red fluorescent f ibers microscopically. Evans blue-stained muscle fibers were either hy percontracted or degrading. Intact or regenerating muscle fibers in md x mice and muscle fibers in B10 control mice were not stained with the dye, DNA isolated from Evans blue-stained fibers exhibited fragmentat ion to approximately 180 base pairs on agarose gel electrophoresis. Su ch DNA fragmentation was not found in DNA from unstained muscle fibers in mdx or B10 mice. Terminal deoxynucleotidyl transferase (TdT)-media ted dUTP nick end-labeling (TUNEL)-positive myonuclei were also found in Evans blue-stained muscle fibers but not in unstained ones, These r esults indicate that degenerating muscle fibers in the mdx mouse show an increase in membrane permeability and undergo apoptosis, Vital stai ning with Evans blue is useful not only for distinguishing degeneratin g muscle fibers, but also for studying the degeneration process bioche mically in dystrophin-deficient muscle, This method may also be useful for evaluating the therapeutic effects of drug administration, gene t ransfer, and myoblast transfer in the mdx mouse.