The D2.mdx mouse as a preclinical model of the skeletal muscle pathology associated with Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle degenerative disease caused by loss of dystrophin protein. DMD has no cure and few treatment options. Preclinical efforts to identify potential DMD therapeutics have been hampered by lack of a small animal model that recapitulates key features of the human disease. While the dystrophin-deficient mdx mouse on the C57BL/10 genetic background (B10.mdx) is mildly affected, a more severe muscle disease is observed when the mdx mutation is crossed onto the DBA/2J genetic background (D2.mdx). In this study, the functional and histological progression of the D2.mdx skeletal muscle pathology was evaluated to determine the distinguishing features of disease. Data herein details the muscular weakness and wasting exhibited by D2.mdx skeletal muscle, as well as severe histopathological features, which include the rapid progression of fibrosis and calcifications in the diaphragm and progressive fibrosis accumulation in limb muscles. Furthermore, a timeline of D2.mdx progression is provided that details distinct stages of disease progression. These data support the D2.mdx as a superior small animal model for DMD, as compared to the B10.mdx model. The insights provided in this report should facilitate the design of preclinical evaluations for potential DMD therapeutics.

Figure 1

Functional comparison of diaphragm and EDL function in B10.mdx and D2.mdx mice. (a) Diaphragm specific tension (SPo) and (b) maximum force production (Po) and (c) SPo of the extensor digitorum longus (EDL) from 4 and 12 month-old B10.mdx and D2.mdx male mice. Values depict muscle function relative to the age- and strain-matched wild-type (WT) mice, and are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Welch’s T-tests (*p < 0.05 vs. B10.mdx values).

Figure 2

Representative histological images from 4 month-old skeletal muscle. Diaphragm and gastrocnemius sections from 4 month-old C57BL/10, DBA/2J, B10.mdx and D2.mdx are presented to display histopathological phenotypes between the mouse lines. Diaphragm muscles displayed are stained with hematoxylin and eosin (H&E), Masson’s trichrome, picrosirius red, and Alizarin red. Gastrocnemius muscles displayed are stained with H&E and Masson’s trichrome. Scale bars represent 100 µm.

Figure 3

Comparison of centrally-located nuclei in B10.mdx and D2.mdx skeletal muscle. Centrally-located nuclei were quantified in the diaphragm and gastrocnemius (Gastroc) muscles of 4 mo B10.mdx and D2.mdx mice (n = 6–14). Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Welch’s T-tests (*p < 0.05 vs. B10.mdx values).

Figure 4

Decalcification of D2.mdx muscle to measure intramuscular fibrosis. Serial sections of a 4 month-old D2.mdx mouse diaphragm were prepared for staining with (a) Alizarin red and picrosirius red to demonstrate prevalence of intramuscular calcifications without decalcification treatment. (b) The third serial section received decalcification treatment prior to picrosirius red staining, allowing accurate histological analysis of intramuscular fibrosis using K-means clustering segmentation analysis in ImageJ software. Scale bar represents 100 µm.

Figure 5

Histological quantification of D2.mdx skeletal muscle fibrosis. Representative picrosirius red and hematoxylin and eosin (H&E) images and fibrosis quantifications for (a,b) diaphragm and (c,d) gastrocnemius muscles from male wild-type (4 month-old DBA/2J) and 4, 6, and 12 month-old D2.mdx mice (n = 6–15). Values are reported as mean ± SEM, and data were analyzed using one-way ANOVA followed by Tukey post-hoc tests (*p ≤ 0.05 vs. wild-type levels; ^#p ≤ 0.05 vs. 4- and 6-month levels).

Figure 6

Histological time course of the development of D2.mdx skeletal muscle pathology. (a) Hematoxylin and eosin (H&E) stained sections of 1 month-old D2.mdx diaphragm and gastrocnemius (Gastroc) exhibiting inflammation without signs of degeneration. (b) Masson’s trichrome staining of 2 month-old samples reveal large degenerating lesions that precede the development of fibrosis. (c) H&E and picrosirius red staining of 3 month-old skeletal muscle showing major histopathological features following recovery from the degenerative stage. (d) Quantification of centrally-located nuclei in the diaphragms and Gastrocs of 3, 4, 6, and 12 mo D2.mdx mice (n = 5–12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. Scale bars represent 100 µm.

Figure 7

Inflammation-associated cytokine gene expression in D2.mdx skeletal muscle. Gene expression of (a) Tnfa, (b) Infg, (c) Il6, (d) Tgfb1, (e) Il10, and (f) Il4 in the diaphragms of 1, 2, 4, and 8 month-old D2.mdx mice (n = 4). Values are quantified relative to those of 4 month-old DBA/2J (WT) samples (n = 4). Values are reported as mean ± SEM, and data were analyzed using one-way ANOVA followed by Tukey post-hoc tests (*p ≤ 0.05 vs. WT levels).

Figure 8

Summary of pathological features of D2.mdx skeletal muscle disease progression. Schematic representation of the progression of dystrophic features in D2.mdx skeletal muscle, including degeneration/inflammation, regeneration, fibrosis, and muscle atrophy.