Impaired regenerative capacity and lower revertant fibre expansion in dystrophin-deficient mdx muscles on DBA/2 background

Figures

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  Figure 1. Mdx-DBA displays a lower percentage of centrally nucleated fibres than mdx-B6. Centrally nucleated fibres were detected by (A) haematoxylin and eosin (H&E) staining and (B) immunostaining with anti-laminin α2 antibody (green) and DAPI nuclear staining (blue). Representative images in tibialis anterior muscles of mdx-DBA and mdx-B6 mice at ages 2, 6, 12 and 18 months are shown. Size reduction of myofibres with age was observed in mdx-DBA mice. Wild-type C57BL/6 muscle at 2 months of age is shown as a control. M: months. Scale bar = 100 μm. (C) The percentage of centrally nucleated fibres per section in H&E staining. Values are represented as mean ± S.D. (n = 4–6 mice per mdx-DBA group). H&E staining results of mdx-B6 mice were obtained from our previous report with permission8. *P < 0.05, **P < 0.01 between mdx-DBA and mdx-B6 mice; †P < 0.05, ††P < 0.01 compared to 6 months old mdx-B6 mice. Statistics: One-Way ANOVA & Tukey-Kramer Multiple Comparisons Test.

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  Figure 2. Nascent regenerating muscle fibres are rarely observed in mdx-DBA. The regenerating fibres in tibialis anterior muscles were detected with anti-developmental myosin heavy chain (MHCd) antibody (red). Nuclei were stained with DAPI (blue). Insets depict zoomed-in MHCd-positive fibres defined by the white box. MHCd-positive fibres were not detected in mdx-DBA at 12 and 18 months of age. Representative images are shown from 4–6 mice in each age group. Wild-type C57BL/6 TA muscle at 2 months old is shown as a negative control. M: months. Scale bar = 100 μm.

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  Figure 3. Immunoglobulin accumulation in mdx-DBA muscles and mdx-B6 muscles. The accumulation of immunoglobulin in tibialis anterior and gastrocnemius muscles was detected by anti-mouse IgG antibody. IgG-positive fibres represent necrotic fibres. Representative images in tibialis anterior muscles are shown from 4–7 mice in each age group of mdx-DBA and mdx-B6. Wild-type C57BL/6 TA muscle at 2 months old is shown as a negative control. M: months. Scale bar = 100 μm.

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  Figure 4. Dystrophin-positive revertant fibres (RFs) at ages 2, 6, 12, and 18 months in mdx-DBA and mdx-B6 mice. Dystrophin signals were detected with an anti-dystrophin C-terminal antibody. Representative images of the maximum RFs in a cluster in tibialis anterior muscles are shown from 3–6 mice in each age group of mdx-DBA and mdx-B6. Mdx-B6 shows higher RF expansion than mdx-DBA in all age groups. Wild-type C57BL/6 TA muscle at 2 months old is shown as a positive control. M: months. Scale bar = 100 μm.

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  Figure 5. Expression and expansion of RFs are lower in mdx-DBA mice compared to mdx-B6 mice in tibialis anterior and gastrocnemius muscles at 2, 6, 12 and 18 months of age. The number of RFs per section was counted according to the following categories: (A) the number of RFs, (B) the number of RF clusters, and (C) the maximum number of RFs in a single cluster. Mdx-B6 mice have a significantly higher RF expression in all categories than mdx-DBA mice. Values are scored as mean ± S.D. (n = 4–6 mdx-DBA mice per each group). Data for mdx-B6 mice were obtained from our previous report with permission8. *P < 0.05, **P < 0.01 between mdx-DBA and mdx-B6 mice; †P < 0.05, ††P < 0.05 compared to 2 months old mdx-B6 mice. M: months. Statistics: One-Way ANOVA & Tukey-Kramer Multiple Comparisons Test.