![]() Recently, Muscle J, a fully automated plug-in in ImageJ described the fully automated quantification of CSA ( 11). A Windows program called MyoVision was developed to fully automate the quantification process, and seems to provide a more accurate CSA measurement compared with SMASH on uninjured muscle ( 10). For example, SMASH is a semiautomated open source MATLAB script allowing the assessment of several parameters, including CSA ( 9). Given the challenges of manual fiber marking, automated programs were developed for measurement of CSA in a whole muscle cross section. This is particularly important when analyzing muscle that is damaged, regenerating, hypertrophying, or diseased. In addition, if the user is interested in the variation and distribution of fiber sizes, many fibers need to be traced, which is challenging to carry out manually. Due to variability of fiber size throughout the section, the most accurate and reproducible results are expected when the entire section is analyzed ( 6). Furthermore, since manual marking of a whole section is generally not feasible due to time requirements, many researchers tend to mark only a portion of the fibers in different parts of the section ( Fig. Such manual marking of muscle fibers is highly time-consuming, subjective, and limits the number of fibers that can be analyzed ( Fig. To measure fiber cross-sectional area (CSA), sections of frozen or paraffin-embedded muscles are generally fluorescently stained with a membrane marker such as wheat germ agglutinin (WGA) ( 8), a lectin which binds glycoconjugates at the membrane, or an antibody against laminin or dystrophin, and images of these sections are subsequently analyzed for CSA measurements by the manual marking of fiber boundaries. Hence, an accurate and efficient measurement of fiber size is important for the study of muscle atrophy, hypertrophy, regeneration, and disease. In addition, in regenerating ( 6) or diseased (e.g., Duchenne muscular dystrophy) ( 7) muscles, there is an increase in fiber size variability. ![]() A reduction in muscle fiber size (i.e., atrophy) occurs in various catabolic states including fasting, denervation, immobilization ( 1), type-2 diabetes, cancer, renal failure ( 2), and aging ( 3), whereas an increase in fiber size (i.e., hypertrophy, growth) is induced by resistance training ( 4), insulin or insulin-like growth factor 1 (IGF-1) treatment, and myostatin deletion ( 5). Skeletal muscle size is affected by a wide variety of physiological and pathological conditions. As a novel addition to the commonly used statistics, we also describe statistical tests that quantify the strength of an effect on fiber size, enabling detection of significant differences between skewed distributions that would otherwise not be detected using typical methods. The Imaris semiautomated approach is user-friendly, requires little training or optimization, and can be used to efficiently and accurately mark thousands of fibers in a short period. ![]() Although both approaches seem valid for measurements of muscle fiber size, the manual marking method is less preferable because it is highly time-consuming, subjective, and limits the number of cells that can be analyzed. Accordingly, both the Imaris semiautomated and manual approaches showed a similar increase in CSA of fibers expressing calpain-1 shRNA compared with adjacent nontransfected fibers in the same muscle cross section. In addition, we previously demonstrated that downregulation of the Ca 2+-specific protease calpain-1 attenuates muscle atrophy. Analysis of muscle cross sections by the Imaris semiautomated and manual approaches demonstrated a similar decrease in CSA of atrophying muscles from fasted mice compared with fed controls. ![]() We adapted the Imaris software to automatically segment muscle fibers based on fluorescent labeling of the plasma membrane and measure muscle fiber CSA. Hence, muscle fiber cross-sectional area (CSA) is an important determinant of muscle health and plasticity. The size and shape of skeletal muscle fibers are affected by various physiological and pathological conditions, such as muscle atrophy, hypertrophy, regeneration, and dystrophies. ![]()
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