Abstract
BackgroundHistological assessment of skeletal muscle sections is important for the research of muscle physiology and diseases. Quantifiable measures of skeletal muscle often include mean fiber diameter, fiber size distribution, and centrally nucleated muscle fibers. These parameters offer insights into the dynamic adaptation of skeletal muscle cells during repeated cycles of degeneration and regeneration associated with many muscle diseases and injuries. Computational programs designed to obtain these parameters would greatly facilitate such efforts and offer significant advantage over manual image analysis, which is very labor-intensive and often subjective. Here, we describe a customized pipeline termed MuscleAnalyzer for muscle histology analysis based upon CellProfiler, a free, open-source software for measuring and analyzing cell images.ResultsThe MuscleAnalyzer pipeline consists of loading, adjusting, and running a series of image-processing modules provided by CellProfiler. This pipeline was evaluated using wild-type and mdx muscle sections co-stained with laminin (to demarcate the muscle fiber boundaries) and 4′,6-diamidino-2-phenylindole (DAPI, to label the nuclei). The immunofluorescence images analyzed using the MuscleAnalyzer pipeline or manually yielded similar results in the number of muscle fibers per image (p = 0.42) and central nucleated fiber (CNF) percentage (p = 0.29) in mdx mice. However, for a total of 67 images, CellProfiler completed the analysis in ~ 10 min on a regular PC while it took an investigator ~ 3 h using the manual approach in order to quantify the number of muscle fibers and CNF. Moreover, the MuscleAnalyzer pipeline also provided the measurement of the cross-sectional area (CSA) and minimal Feret’s diameter (MFD) of muscle fibers, and thus fiber size distribution can be plotted.ConclusionsOur data indicate that the MuscleAnalyzer pipeline can efficiently and accurately analyze laminin and DAPI co-stained muscle images in a batch format and provide quantitative measurements for muscle histological properties such as muscle fiber diameters, fiber size distribution, and CNF percentage.
Highlights
Histological assessment of skeletal muscle sections is important for the research of muscle physiology and diseases
Muscle fiber and nuclei identification The laminin α2 and DAPI co-stained muscle sections of WT and mdx mice were imaged and saved to ND2 files using the NIS-Elements Advanced Research software provided by Nikon (Fig. 1)
CellProfiler supports a wide variety of image formats, including most of those used in imaging, by using a library called Bio-Formats
Summary
Histological assessment of skeletal muscle sections is important for the research of muscle physiology and diseases. Quantifiable measures of skeletal muscle often include mean fiber diameter, fiber size distribution, and centrally nucleated muscle fibers These parameters offer insights into the dynamic adaptation of skeletal muscle cells during repeated cycles of degeneration and regeneration associated with many muscle diseases and injuries. Satellite cells associated with skeletal muscle are activated to proliferate, fuse to form myotubes, and eventually regenerate new muscle fibers [4,5,6,7] In genetic myopathies such as Duchenne muscular dystrophy (DMD), a fatal X-linked recessive muscle disease caused by genetic mutations leading to the loss of dystrophin [8], repeated cycles of muscle injury, and repair result in increased variation of fiber size and muscle fibers with central nuclei [9, 10].
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