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. 2020 Oct 12;10:16385. doi: 10.1038/s41598-020-73315-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Progressive exacerbation of muscular dystrophy in DMD rats. (a) Representative images of 6-month-old WT (left panel) and DMD rats (right panel). (b) Body weight comparison of WT and DMD rats of 1–9 months of age (n = 6). (c) Quantification of maximum muscle strength by grip test at the indicated ages of WT and DMD rats (2 months: WT: n = 7, DMD: n = 6, 4 months: WT: n = 7, DMD: n = 7, 6 months: WT: n = 8, DMD: n = 12, 8 months: WT: n = 4, DMD: n = 7, 10 months: WT: n = 11, DMD: n = 21). (d) Representative HE stains of TA muscle sections from 6-month-old WT rats and 1- to 10-month-old DMD rats. Scale bar = 100 μm. The following symbols, ψ, *, #, and ζ, indicate inflammatory cell infiltration, necrotic myofibres, fibrosis, and adipogenesis, respectively. (e) Immunoblotting analysis of perilipin expression in WT and DMD rats. Full-length blots are presented in Supplementary Figure 6a. (f) Quantification of perilipin protein expression (n = 6, each). (g) Representative Masson Trichrome stains of TA muscle sections from 1- to 10-month-old WT and DMD rats. Scale bar = 100 μm. (h) Quantification of Masson Trichrome staining positive area per section (n = 6, each). (i) Immunohistochemical analysis of eMHC in TA muscle sections from WT and DMD rats. Scale bar = 100 μm. (j) Quantification of eMHC positive fibres per section (n = 6, each). (k,l) Quantification of (k) Pax7⁺ cells and (l) MyoD⁺ cells of skeletal muscle primary cells from WT and DMD rats (n = 6, each). Data are expressed as mean ± SEM except for (b), which is expressed as mean ± SD, and were compared by Tukey Kramer’s test. Different letters indicate statistically significant differences (p < 0.05). Progressive decrease of both Pax7⁺ and MyoD⁺ cells was observed in DMD rats. Significant decrease of the number of MyoD⁺ cells was observed in DMD rats from 1 month compared to WT, while from 3 months about the number of Pax7⁺ cells. For (c), (h), (k) and (l), the result of statistical comparison only between the genotypes at each indicated ages was displayed. When a significant age-related difference was observed by the Tukey–Kramer’s test, the † mark was added beside the legend of the graph. *p < 0.05. **p < 0.01. ***p < 0.001. N.D. not detected.