{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T18:25:46Z","timestamp":1793039146330,"version":"4.5.0"},"reference-count":38,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,2,22]],"date-time":"2022-02-22T00:00:00Z","timestamp":1645488000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100007393","name":"French Muscular Dystrophy Association","doi-asserted-by":"publisher","award":["22613"],"award-info":[{"award-number":["22613"]}],"id":[{"id":"10.13039\/100007393","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Adult skeletal muscle is capable of active and efficient differentiation in the event of injury in both physiological and pathological conditions, such as in Duchenne muscular dystrophy (DMD). DMD is characterized by different features, such as continuous cycles of degeneration\/regeneration, fiber heterogeneity, chronic inflammation and fibrosis. A well-defined and standardized approach for histological and morphometric analysis of muscle samples is necessary in order to measure and quantify specific regenerative parameters in myopathies. Indeed, non-automatic methods are time-consuming and prone to error. Here, we describe a simple automatized computational approach to quantify muscle parameters with specific pipelines to be run by CellProfiler software in an open-source and well-defined fashion. Our pipelines consist of running image-processing modules in CellProfiler with the aim of quantifying different histopathological muscle hallmarks in mdx mice compared to their wild-type littermates. Specifically, we quantified the minimum Feret diameter, centrally nucleated fibers and the number of macrophages, starting from multiple images. Finally, for extracellular matrix quantification, we used Sirius red staining. Collectively, we developed reliable and easy-to-use pipelines that automatically measure parameters of muscle histology, useful for research in myobiology. These findings should simplify and shorten the time needed for the quantification of muscle histological properties, avoiding challenging manual procedures.<\/jats:p>","DOI":"10.3390\/diagnostics12030561","type":"journal-article","created":{"date-parts":[[2022,2,22]],"date-time":"2022-02-22T22:35:09Z","timestamp":1645569309000},"page":"561","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0726-073X","authenticated-orcid":false,"given":"Valerio","family":"Laghi","sequence":"first","affiliation":[{"name":"Unit\u00e9 Macrophages et D\u00e9veloppement de l\u2019Immunit\u00e9, Institut Pasteur, CNRS UMR3738, 75015 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9671-2659","authenticated-orcid":false,"given":"Valentina","family":"Ricci","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Cell Biology (IBBC), National Research Council of Italy (CNR), Via Ercole Ramarini, 32, Monterotondo, 00015 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0034-5450","authenticated-orcid":false,"given":"Francesca","family":"De Santa","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Cell Biology (IBBC), National Research Council of Italy (CNR), Via Ercole Ramarini, 32, Monterotondo, 00015 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9436-8783","authenticated-orcid":false,"given":"Alessio","family":"Torcinaro","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Cell Biology (IBBC), National Research Council of Italy (CNR), Via Ercole Ramarini, 32, Monterotondo, 00015 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,22]]},"reference":[{"key":"ref_1","first-page":"1984879","article-title":"Current Methods for Skeletal Muscle Tissue Repair and Regeneration","volume":"2018","author":"Liu","year":"2018","journal-title":"Biomed Res. Int."},{"key":"ref_2","first-page":"821950","article-title":"Inflammation in Muscle Repair, Aging, and Myopathies","volume":"2014","author":"Rossi","year":"2014","journal-title":"Biomed Res. Int."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Madaro, L., Torcinaro, A., De Bardi, M., Contino, F.F., Pelizzola, M., Diaferia, G.R., Imeneo, G., Bouch\u00e9, M., Puri, P.L., and De Santa, F. (2019). Macrophages Fine Tune Satellite Cell Fate in Dystrophic Skeletal Muscle of Mdx Mice. PLoS Genet., 15.","DOI":"10.1371\/journal.pgen.1008408"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1016\/0092-8674(87)90579-4","article-title":"Dystrophin: The Protein Product of the Duchenne Muscular Dystrophy Locus","volume":"51","author":"Hoffman","year":"1987","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.1073\/pnas.81.4.1189","article-title":"X chromosome-linked muscular dystrophy (mdx) in the mouse","volume":"81","author":"Bulfield","year":"1984","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Gutpell, K.M., Hrinivich, W.T., and Hoffman, L.M. (2015). Skeletal Muscle Fibrosis in the Mdx\/Utrn +\/\u2212 Mouse Validates Its Suitability as a Murine Model of Duchenne Muscular Dystrophy. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0117306"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e12391","DOI":"10.14814\/phy2.12391","article-title":"Disease Course in Mdx:Utrophin +\/\u2212 Mice: Comparison of Three Mouse Models of Duchenne Muscular Dystrophy","volume":"3","author":"Mcdonald","year":"2015","journal-title":"Physiol. Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1093\/hmg\/11.3.263","article-title":"A Chronic Inflammatory Response Dominates the Skeletal Muscle Molecular Signature in Dystrophin-Deficient Mdx Mice","volume":"11","author":"Porter","year":"2002","journal-title":"Hum. Mol. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1186\/2044-5040-1-21","article-title":"Aberrant Repair and Fibrosis Development in Skeletal Muscle","volume":"1","author":"Mann","year":"2011","journal-title":"Skelet. Muscle"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1186\/s13395-018-0186-6","article-title":"Open-CSAM, a New Tool for Semi-Automated Analysis of Myofiber Cross-Sectional Area in Regenerating Adult Skeletal Muscle","volume":"9","author":"Desgeorges","year":"2019","journal-title":"Skelet. Muscle"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Henry, C.C., Martin, K.S., Ward, B.B., Handsfield, G.G., Peirce, M., and Blemker, S.S. (2017). Spatial and Age-Related Changes in the Microstructure of Dystrophic and Healthy Diaphragms. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0183853"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1002\/cyto.a.20334","article-title":"Fully Automated Segmentation and Morphometrical Analysis of Muscle Fiber","volume":"71","author":"Kim","year":"2007","journal-title":"Cytometry"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/B978-0-12-407694-5.00007-9","article-title":"Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type","volume":"306","author":"Kostrominova","year":"2013","journal-title":"Int. Rev. Cell Mol. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Pertl, C., Eblenkamp, M., Pertl, A., Pfeifer, S., Wintermantel, E., Lochm\u00fcller, H., Walter, M.C., Krause, S., and Thirion, C. (2013). A New Web-Based Method for Automated Analysis of Muscle Histology. BMC Musculoskelet. Disord., 14.","DOI":"10.1186\/1471-2474-14-26"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1186\/2044-5040-4-21","article-title":"SMASH\u2014Semi-Automatic Muscle Analysis Using Segmentation of Histology: A MATLAB Application","volume":"4","author":"Smith","year":"2014","journal-title":"Skelet. Muscle"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.compbiomed.2015.04.020","article-title":"Automated high-content morphological analysis of muscle fiber histology","volume":"63","author":"Miazaki","year":"2015","journal-title":"Comput. Biol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1186\/s13395-018-0171-0","article-title":"MuscleJ: A High-Content Analysis Method to Study Skeletal Muscle with a New Fiji Tool","volume":"8","author":"Hardy","year":"2018","journal-title":"Skelet. Muscle"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1152\/japplphysiol.00762.2017","article-title":"MyoVision: Software for Automated High-Content Analysis of Skeletal Muscle Immunohistochemistry","volume":"124","author":"Wen","year":"2018","journal-title":"J. Appl. Physiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/978-1-62703-311-4_8","article-title":"CellProfiler and KNIME: Open Source Tools for High Content Screening","volume":"986","author":"Niederlein","year":"2013","journal-title":"Methods Mol. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1186\/gb-2006-7-10-r100","article-title":"CellProfiler: Image Analysis Software for Identifying and Quantifying Cell Phenotypes","volume":"7","author":"Carpenter","year":"2006","journal-title":"Genome Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1093\/bioinformatics\/btr095","article-title":"Improved Structure, Function and Compatibility for CellProfiler: Modular High-Throughput Image Analysis Software","volume":"27","author":"Kamentsky","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Mcquin, C., Goodman, A., Chernyshev, V., Kamentsky, L., Cimini, A., Karhohs, K.W., Doan, M., Ding, L., Rafelski, S.M., and Thirstrup, D. (2018). CellProfiler 3.0: Next-generation image processing for biology. PLoS Biol., 16.","DOI":"10.1371\/journal.pbio.2005970"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Stirling, D.R., Bowden, M.J.S., Lucas, A.M., Carpenter, A.E., Cimini, B.A., and Goodman, A. (2021). CellProfiler 4: Improvements in Speed, Utility and Usability. BMC Bioinform., 22.","DOI":"10.1186\/s12859-021-04344-9"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Torcinaro, A., Ricci, V., Strimpakos, G., De Santa, F., and Middei, S. (2021). Peripheral Nerve Impairment in a Mouse Model of Alzheimer\u2019s Disease. Brain Sci., 11.","DOI":"10.3390\/brainsci11091245"},{"key":"ref_25","first-page":"36","article-title":"Image Processing with ImageJ","volume":"11","author":"Hospitals","year":"2004","journal-title":"Biophotonics Int."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An Open-Source Platform for Biological-Image Analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1109\/TSMC.1979.4310076","article-title":"A Threshold Selection Method from Gray-Level Histograms","volume":"9","author":"Otsu","year":"1979","journal-title":"IEEE Trans. Syst. Man Cybern."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1152\/japplphysiol.01022.2012","article-title":"Automated Image Analysis of Skeletal Muscle Fiber Cross-Sectional Area","volume":"114","author":"Mula","year":"2013","journal-title":"J. Appl. Physiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1016\/0031-3203(93)90115-D","article-title":"Minimum Cross Entropy Thresholding","volume":"26","author":"Li","year":"1993","journal-title":"Pattern Recognit."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1553","DOI":"10.1089\/ars.2017.7420","article-title":"The Role of Metabolic Remodeling in Macrophage Polarization and Its Effect on Skeletal Muscle Regeneration","volume":"30","author":"Vitiello","year":"2019","journal-title":"Antioxid. Redox Signal."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1007\/978-1-4939-1215-5_16","article-title":"Generation of transgenic mouse fluorescent reporter lines for studying hematopoietic development","volume":"1194","author":"Vacaru","year":"2014","journal-title":"Methods Mol. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/s13395-018-0178-6","article-title":"Automated Muscle Histopathology Analysis Using CellProfiler","volume":"8","author":"Lau","year":"2018","journal-title":"Skelet. Muscle"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1016\/j.nmd.2004.06.008","article-title":"Histological Parameters for the Quantitative Assessment of Muscular Dystrophy in the Mdx-Mouse","volume":"14","author":"Briguet","year":"2004","journal-title":"Neuromuscul. Disord."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1698","DOI":"10.1152\/japplphysiol.00257.2019","article-title":"Muscle2View, a CellProfiler Pipeline for Detection of the Capillary-to-Muscle Fiber Interface and High-Content Quantification of Fiber Type-Specific Histology","volume":"127","author":"Sanz","year":"2019","journal-title":"J. Appl. Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1016\/S0140-6736(17)32160-8","article-title":"Long-Term Effects of Glucocorticoids on Function, Quality of Life, and Survival in Patients with Duchenne Muscular Dystrophy: A Prospective Cohort Study","volume":"391","author":"Mcdonald","year":"1989","journal-title":"Lancet"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e202000646","DOI":"10.26508\/lsa.202000660","article-title":"Metabolic reprogramming of fibro\/adipogenic progenitors facilitates muscle regeneration","volume":"3","author":"Reggio","year":"2020","journal-title":"Life Sci. Alliance"},{"key":"ref_37","first-page":"315","article-title":"Wheat Germ Agglutinin Staining as a Suitable Method for Detection and Quantification of Fibrosis in Cardiac Tissue after Myocardial Infarction","volume":"58","author":"Emde","year":"2014","journal-title":"Eur. J. Histochem."},{"key":"ref_38","first-page":"570","article-title":"Labeling Membrane Glycoproteins or Glycolipids with Fluorescent Wheat Germ Agglutinin","volume":"5","author":"Chazotte","year":"2011","journal-title":"Cold Spring Harb. Protoc."}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/12\/3\/561\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:25:04Z","timestamp":1760135104000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/12\/3\/561"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,22]]},"references-count":38,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,3]]}},"alternative-id":["diagnostics12030561"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics12030561","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,22]]}}}