These macrophages phagocyte muscle debris and promote the proliferation of muscle stem cells. Just after injury, monocytes infiltrate the tissue from the blood and convert into pro-inflammatory damaged associated macrophages. Inflammation, and notably macrophages, plays important roles during this process.
Skeletal muscle regenerates ad integrum after a sterile acute injury thanks to satellite cells (muscle stem cells). The online version of this article (10.1186/s1339-6) contains supplementary material, which is available to authorized users. Our 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. 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.
CELLPROFILER 2.2.0 MANUAL
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. 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. 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 MuscleAnalyzer pipeline consists of loading, adjusting, and running a series of image-processing modules provided by CellProfiler.
CELLPROFILER 2.2.0 SOFTWARE
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. 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. 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. Quantifiable measures of skeletal muscle often include mean fiber diameter, fiber size distribution, and centrally nucleated muscle fibers. Histological assessment of skeletal muscle sections is important for the research of muscle physiology and diseases.
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